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Fertility Symposium

Ken Seethram, MD, FRCSC, FACOG

Pacific Centre for Reproductive Medicine

Clinical Assistant Professor, UBC & University of Alberta

kseethram@pacificfertility.ca

Prenatal Screening

All that you need to know…..

Outline• To understand

• Prenatal Screening and amniocentesis/CVS

• Non-Invasive Prenatal Testing/Screening

• To understand the scope of NIPT and its variations

• To understand the detection rates and limitations of NIPT

• To understand the various forms of prenatal screening, and who gets what in BC

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Disclosure• I have no financial interest in the current NIPT providers – we receive

no research grants, educational grants, or other initiatives from any NIPT provider

• We draw and send plasma on patients for NIPT in our centre

• We perform first trimester screening (non-insured service) in our centre

• Warning: trade names will be used during this talk

Prenatal Screening &

Diagnosis for Chromosome Abnormalities

• T21 is one of the most common aneuploidy to affect live-born children

and has a background prevalence of 1:691, increasing with maternal age

• What causes T21 – hypo-methylation by the extra chromosome

• Prenatal diagnosis relies upon the procurement of fetal cells via amniocentesis (ACT) / trophoblast via chorionic villus sampling (CVS)

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1977-That would have been the end of this talk

Prenatal Screening & Diagnosis for Chromosome Abnormalities

• Simpler times back then; if your age based risk exceeded the risk of diagnostic testing, then do the test (which was

roughly age 35-38)

• So the only screening tool we had was age

• What is the risk of Amniocentesis or CVS?

• For the last 40 years, it has been quoted at 1%

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What is the RISK of CVS/ACT• A recent meta-analysis (Akolekar, et al (2015) Procedure-related risk of miscarriage following

amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound in Obstetrics and Gynecology 45(1):16):

• 42,000 women who underwent ACT and 138,000 who did not, data collected >1999

• Attributable miscarriage risk: 0.11%

• 54,000 women who underwent CVS and 670,000 who did not, data collected >1999

• Attributable miscarriage risk: 0.22%

• Odibo, et al (2008) Revisiting the fetal loss rate after second-trimester genetic amniocentesis: a single center's 16-year experience. Obstetrics and Gynecology 11(3):589

• 11 746 amniocenteses and 5243 CVS

• 0.13% loss rate in amnio and 0.7% loss rate in CVS was no different than the loss rate in those without invasive procedures

• Our previously agreed-upon 1% may not be so true

Invasive Testing• The risks of invasive testing should govern the decision to

do the test.

• However….end of the day – nobody wants invasive testing even if the risk of loss is 1/1000

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What is the point of prenatal screening?

• To reduce the risk of invasive testing

• by limiting the invasive test to those most in

need (the highest risk people)

Prenatal screening• And How?

• �Age

• Double screening>Triple marker screening>Quad Screening

• MSAFP for Neural tube defect screening in the early 80’s was then linked with hCG for T21 detection

• 1991 (Cuckle and Wald) published in the BJOB – uE3, hCG for T21 detection

• Combined this became triple marker screening

• Modified in 1996 by the addition of dimeric Inhibin-A to become QUAD

screen

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Prenatal screening• And How?

• �Age

• � Double screening>Triple marker screening>Quad Screening

• First Trimester Combined Screening (NT+NB+DV+PAPP-A+bHCG)

• BMJ 1992 – K. Nicolaides published on the use of beta-hCG, PAPP-A and a single ultrasound measurement at 12w called nuchal translucency (NT)

• Modified since 1992 to include nasal bone, FHR, ductus venosus flow (hepatic vein flow) using color flow Doppler measurements

• Accreditation provided through single site – Fetal Medicine Foundation, UK

• Detection rate: 96% with screen positive rate = 3%

Prenatal screening• And How?

• �Age

• � Double screening>Triple marker screening>Quad Screening

• � First Trimester Combined Screening (NT+NB+DV+PAPP-A+bHCG)

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Prenatal screening• By the end of 2009, there were really two camps in

prenatal screening

Prenatal screening

First Trimester People•Ultrasound based

•Early

•Dates

•Twins etc

•Anomaly screening

Pleasers•PAPP-A + Quad (SIPS)

•NT + PAPP-A + QUAD (IPS)

Second Trimester People•Serum based

•Placental screening (QUAD)

DR 96%

SRP 3%

DR 88%

SRP 3% DR 75%

SRP 5%

100

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Prenatal screening• And How?

• � Age

• � Double screening>Triple marker screening>Quad Screening

• � Combined First Trimester Screening

• And then things got really complicated in 2011…..NIPT

NIPT – ability to detect ‘fetal’ DNA • Emerged in 2011, but first described in 1997

• “cell-free ‘fetal’ DNA (cffDNA),” actually derived from the placenta=4% of all free-DNA in the maternal blood

• Can be detected as early as 4-5 weeks of gestation

• Usually does not exceed 150 base pairs of length (very small fragments) but the entire fetal genome is represented

• Keep in mind:• Our entire genome - 6.5B base pairs• Chromosome 1 has 249M base pairs• Chromosome 21 has 48M base pairs• Chromosome 22 has 49.5M base pairs

• So there’s a lot of analysis required to re-construct part or all of the fetal genome from these bits of DNA

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Human Genome Project in the meantime…

Human Genome Project and NIPT• Fragments of DNA can then be compared against library’s

like Venter’s Human Genome Library (HuRef) to

essentially either sequence the genome from those fragments, or use targeted gene analysis to count signals

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NIPT• How does it work?

• In aneuploidy, the placenta is also aneuploid, therein releasing

more DNA of particular chromosomes versus the balanced number expected – this principle is the basis for NIPT

• Blood draw – timing depends on the product, but

generally as early as 9w.

Methods – NIPT (1997-2015)• 1997 First paper published demonstrating the presence of cell free fetal

DNA. (PCR)

• 2008 Use of s-MPS to identify millions of fragments of DNA and their specific chromosome origin. This is whole genome sequencing (MaterniT21)• Very costly, and timely, requiring up to 10M base pairs to be sequenced

• 2009-2010 – introduction of targeted sequencing for identification of set loci • on certain chromosomes (Harmony)

• 2012 -SNP array (Panorama) – generating a virtual karyotype by determining the copy number of each SNP on the array and aligning the SNP’s in chromosomal order

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Types of NIPT

NIPTNIPT

S-MPSS-MPS

TargetedTargetedSNP ArraySNP Array

Whole Genome

ex. MaterniT21

HarmonyPanorama

NIPT• To clarify some misnomers:

• It’s not non-invasive (although, akin to every other mode of

screening, the fetal risk is zero)

• It’s not testing or diagnosis (although up to 7% of women will

proceed with termination after a positive NIPT without diagnostic confirmation)

• It’s not cell free fetal DNA – it’s placental DNA

• It’s not pronounced NIP-TEE

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NIPT• How accurate is it?

• For T21 - >99.2% with 0.1% screen positive rate

• What is the biggest error?

• Non Reporting• of all the published studies, the rate ranges from 1%-10% and 32%

of the time, when you read a second sample, you’ll still not get

enough fetal fraction to provide analysis

• On average, 3-4% of samples are non reported

Targeted Sequencing (Harmony)

• 99.9% specificity with 0.103% FPR

• Looking for specific genes with 13/18/21 loci and counting their DNA

load relative to genes from other chromosomes

• Not as great for T13 and T18 (about 90%), but for T21 it’s excellent

• <4.0% = low fetal fraction – non reporting risk

• But it’s less expensive than MPS, and excellent for T21

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NIPT using SNP array (Panorama)

• Can provide information about the parent of origin of aneuploidy,

recombination and inheritance of mutations

• Can provide information on a subset of other chromosomal rearrangements such as Angelman syndrome, Prader-Willi, DiGeorgesyndrome

• Can use lower fetal fraction (2.8%) – therefore theoretically less non-reporting

• May flag triploidy or vanishing twins

NIPT using whole genome• Massively parallel sequencing based (BGI, Beijing

Genomics Institute) H. Zhang, 2015

• 147,314 samples

• Sensitivities:

• T21 99.17%

• T18 98.24%

• T13 100%

• Human Genome project – 1988-2003 = 2.7B

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What NIPT is commercially available?

• Ariosa (Roche) Harmony

• Verinata Verifi• Sequenom MaterniT21 Plus

• NIPT plus • 22q deletion syndrome (DiGeorge)• 5p (Cri-du-chat syndrome)

• 15q (Prader-Willi/Angelman syndromes)

• 1p36 deletion syndrome

• 4p (Wolf-Hirschhorn syndrome)• 8q (Langer-Giedion syndrome)

• 11q (Jacobsen syndrome)

• Trisomy 16• Trisomy 22

• NIPT alone VisibiliT

• Genome Wide Maternit GENOME

• Labcorp – Integrated Genetics Informaseq• Natera – NIPT plus microdeletions Panorama

• BGI NIFTY• Berry Genomics BambiniTest

• Premaitha IONA test

• NIPD Genetics Veracity

Which NIPT is best?• The best test is the one which is accurate, cost effective,

and broad in perspective

• Frankly all of them are superb in detection rates

• But…here’s the problem……NIPT is being sold as

prenatal screening – it’s not

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NIPT IS NOT Prenatal Screening• NIPT = Down syndrome screening.

• While we can screen with NIPT less well for T18, T13, 45X, and a smattering of rare disorders, NIPT is T21 screening

• There are some ethical drawbacks to this:

• We might ‘normalize’ testing and termination

• We might invest less in support for T21 children and families

• The billions spent from genomics companies on development and marketing crush the patient’s understanding of the test

• NIPT is another part of the prenatal screen

• It’s also not cytogenetics so should never replace

CVS/ACT

NIPT IS PART of the prenatal screen• And all our guidelines say the same:

• SOGC (updated guideline Jan 2017)• Recommend a 11-14w scan regardless of NIPT• offer NIPT to high risk individuals• Positive tests should always be followed by amniocentesis• No evidence to replace current screening

• ACOG-SMFM• Discuss NIPT and offer it, but as an adjunct to existing screening• Follow up positives with cytogenetics• If markers are seen on ultrasound, it should not substitute CVS/ACT

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NIPT – ISPD guidelines summary

• There is value in adding NIPT to first

trimester screening and/or second trimester quad markers for both

aneuploidy and other prenatal risk

assessment

Why should we not rely on NIPT alone?

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Why should we not rely on NIPT alone?• Conventional screening, whether it’s FTS, or Integrated

screening provides other information including fetal structures, multiples etc.• Baer 2014 ACOG identified 9,051 FTS screen-positive and 30,928

screen-negative pregnancies

• FTS screen positive women were

• 1.7X more likely to be diagnosed with preeclampsia, placenta previa, or abruption

• 3.5X more likely to experience fetal loss before 20 weeks

• Women with positive results for more than one screened condition were at substantially greater risk (34-157X) relative risk of fetal and neonatal mortality

Why should we not rely on NIPT alone?• Baer and Norton AJOG 2014

• Examined NT alone – any measurement over 95%ile was linked with:

• RR of 1.6X for having more than one major structural birth defect, particularly GU, hydrocephalus, lung hypoplasia/agenesis, atresia and stenosis of the small bowel, diaphragm abnormalities

• So elevated NT alone should make us thing of other things, not just aneuploidy

• Norton 2014• 16.9% of FTS screen positive women will have a chromosomal anomaly

which would not be detected by NIPT

• Pergament 2012• Up to 30% of all positive FTS is secondary to aneuploidy not detectable by

NIPT

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So how does NIPT fit within the system?

• NIPT (being another screening tool) has various options to integrate into the already existing system of screening

• Hui and Bianchi (2017) Noninvasive Prenatal DNA Testing:

The Vanguard of Genomic Medicine. Annual Review of

Medicine 68:21.1–21.14.

�Advanced Test Screening

�Universal Screening

�Contingent Model

NIPT Advanced Test ScreeningFirst Trimester Combined Screen

No Further Testing

Low risk Result High Risk Result

Offer NIPT as an advanced

screen

Diagnostic Testing

�Negative?

-reduction in invasive

testing rates

-UK National

Screening Committee recommends

introduction of this

model into NHS

+

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NIPT Universal ScreeningNIPT at 10-12w

(retain serum for FTS)

11-13w ultrasound

(NT)

Normal US and low risk

NIPT

No further testing

Abnormal US and/or high risk NIPT

Diagnostic Testing

‘no-call’ NIPT

Perform Combined

FTS

• most expensive

• provides

maximal

detection for fetal aneuploidy

• early detection

of structural anomalies

NIPT Contingent Model

Combined FTS

Low Risk (<1:1000)

Intermediate Risk (between 1:100

and 1:1000)

NIPT

No Further Testing Diagnostic Testing

High Risk (>1:100)

- +

• Cheaper than the

Universal model,

but better than the

advanced model because more

women access the

NIPT• Ontario moving to

this

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NIPT - FMF Blood draw 10w

Combined FTS

Integrate results

Normal FTS/NIPTAbnormal FTS

or NIPT

ACT/CVS

Abnormal NT beyond 3.5mm

ACT/CVS with array

Abnormal DV

Fetal echo 22w

Serum for PAPP-A/fb-hCG Plasma for NIPT

NIPT• So we’ve learned that NIPT isn’t a stand-alone test

• And we’ve learned that it should be integrated into

existing testing

• What about some special situations?

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Combined FTS special situations• If the NT is over 3.5mm with normal NIPT, diagnostic

testing with microarray is recommended for the detection

of subchromosomal abnormalities – 6% (deletion/duplication sequences)

• If the DV is abnormal, fetal echocardiography is recommended to evaluate CHD

Apart from Aneuploidy, what else does NIPT give us?

• 1. Sex Chromosome aneuploidy

• DR for 45X is 90.3% (compared with 99.2% for T21)

• Keep in mind that as age increases, there can be a somatic loss of one X chromosome in the blood, leading to low-level mosaicism –

leading to false positive results

• Also – confined placental mosaicism (CPM) – for monosomy X is

common (59% on CVS)

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Apart from Aneuploidy, what else does NIPT give us?

• 2. Subchromosomal - (microdeletions/duplications)

• Microdeletion testing now offered by many NIPT include:

1. 5p- (Cri du Chat)

2. 22q11.2- (DiGeorge syndrome)

3. 15q- (Prader-Willi/Angelman syndrome)

4. 4p- (Wolf-Hirschhorn syndrome)

5. 11q- (Jacobsen syndrome)

6. 8q- (Langer-Giedion syndrome)

7. 1p36-

• Keep in mind: these are RARE (ex. 5p- - 1:20,000)

Apart from Aneuploidy, what else does NIPT give us?

• 2. Subchromosomal - (microdeletions/duplications)• Keep in mind that 1.7% of all normal G-band karyotyped fetuses, will

have a copy number variant (CNV)

• NIPT misses 17% of the total cytogenetic abnormalities detected by diagnostic testing

• SMFM and ACOG both state “cell free DNA screening tests for microdeletions have not been validated clinically and are not recommended at this time”

• CNV’s are not related to age

• Therefore at this point, doing NIPT for CNV’s isn’t a standard

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NIPT and special situations• Fetal sex information – China/India

• Donor egg – (targeted)- Harmony

• Vanishing twin – NIPT not indicated

• Triploidy – only post-hoc

• Panorama – no twins

• Single Gene disorders (future)

• AD conditions that are paternally inherited or de novo

• Torsion dystonia, achondroplasia, beta thal, X linked conditions

• May 2017 – Vistara (Natera) -30 gene panel

Screening and direct marketing

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Human Genome Project

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Beware the Direct Marketing• NIPT companies have unlimited access to money to promote their products

• equate DS with developmental delay

• Normal development, learning disability, and autism are top tier issues for expectant parents

• Ergo NIPT must be the right thing to do?

• Direct access to NIPT may be altering our ability to provide more comprehensive screening and counseling

• NIPT is screening – but it’s the first prenatal genetic screening tool which is being directly marketed, removing health care practitioners from the ability to counsel

• Genetic counseling is more vital and paramount than ever before – to assist patients in navigating through all this information.

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Bottom line for NIPT• Doing and recommending NIPT alone is not suggested

• NIPT is an adjunct to existing screening

• NIPT’s great for Down Syndrome, less good for other things

• If you do NIPT alone, you’ll miss up to 30% of other genetic and developmental disorders

• Beware the marketing – screening for microdeletion panels with an incidence of 1:20000 is missing the forest

Options in BC

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Prenatal Screening

First Trimester People•Ultrasound based

•Early

•Dates

•Twins etc

•Anomaly screening

Pleasers•Combining PAPP-A with Quad (SIPS)

•Combining NT, PAPP-A with QUAD (IPS)

Second Trimester People•Serum based

•Placental screening (QUAD)

DR 96%

SRP 3%

DR 88%

SRP 3% DR 75%

SRP 5%

Options in BC – MSP insured

<35 SIPS or QUAD 20W US

35-39 IPS 20W US

>40 IPS orSIPS or

CVS/ACT

20W US

Personal/family history of increased aneuploidy risk for T21/T13/T18

IPS oror

CVS/ACT

20W US

Twins IPS orSIPS or

CVS/ACT

20W US

Pregnancy after ICSI IPS orSIPS or

CVS/ACT

20W US

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Insured NIPT in BC – indications:1. Women with positive IPS/SIPS/Quad

2. Prior T21/18/13

3. Risk >1:300 on QUAD and US aneuploidy marker

Self Pay options in BC• First Trimester Screening (NT, Nasal Bone, Ductus Venosus, PAPP-A,

HCG) - 550

• NIPT

• PCRM 650 Harmony, Panorama, in house counseling

• Olive 650 Harmony, in house counseling

• Grace 600 Harmony, out source counseling

• Lifelabs 550 Panorama basic aneuploidy

745 basic plus 22q11.2 deletion

745 basic plus microdeletion panel

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What about the type-A patient?• Do both: the Universal/FMF model of First trimester

screening plus NIPT

• If normal NIPT and Normal FTS – risk very low

• If normal NIPT, but increased NT>3.5mm – amnio with array

• If normal NIPT but abnormal DV – fetal echo

• Anatomic anomaly or NIPT abnormal - diagnostics