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Cell free fetal DNA testing Implementation into clinical practice Lucy Jenkins, North East Thames Regional Genetics

Cell free fetal DNA testing Implementation into clinical ... · NIPT / NIPS Testing ... – Requires accurate quantification of fetal fraction in cell ... NIPD for recessive conditions

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Cell free fetal DNA testing Implementation into clinical practice

Lucy Jenkins, North East Thames Regional Genetics

NIPD Diagnosis – targeted analysis monogenic disorders

No invasive confirmation required

NIPT / NIPS Testing - Screening for aneuploidy

Requires invasive testing for confirmation

Cell Free Fetal DNA Testing

Identification of alleles that are not present in the mother:

• Identification of RHD+ve pregnancies in RHD –ve mothers

• Fetal sex determination

o detection of Y markers in pregnancies at high genetic risk (X-linked disorders, CAH)

o UKGTN Approval 2011

• Paternally inherited dominant conditions

• De novo gene changes e.g. achondroplasia

• Recessively inherited disease where parents carry different mutations, paternal exclusion

In development

• Recessive conditions where parents carry same mutations

• Detecting inheritance of a maternal mutation against high maternal background DNA

NIPD - Monogenic disorders

Chitty et al. Prenatal Diagnosis 2015 epub

NIPD for skeletal dysplasia - FGFR3 Panel • 29 mutations in 5 FGFR3 amplicons

• ACH, TD

• Next Generation Sequencing – Illumina MiSeq

• Digital read out

TD: >50 tested

100% correct

c.742C>T p.(Arg248Cys)

c.746C>G p.(Ser249Cys)

c.1948A>G p.(Lys650Glu)

c.1118A>G p.(Tyr373Cys)

c.2419T>A p.(*807Argext*101)

No inconclusives

ACH: >80 tested

1 False Negative

(Rare mutation now added to panel)

No inconclusives

NIPD – De Novo Mutations

Benefits of NIPD - thanatophoric dysplasia

NIPD allows risk free early definitive diagnosis from 13/40

Excludes differential diagnoses that may carry high recurrence risk (e.g.SRPS)

Allows for a surgical termination if requested

Provides accurate diagnosis in twins without putting normal twin at risk

Safe, early exclusion of a recurrence in future pregnancies

NIPD FGFR3 Panel UKGTN approval 2013

Only applicable to couples - who are known carriers of different CF mutations AND - the paternal mutation is one of the 11 mutations listed

below

5 CFTR amplicons for MiSeq (Illumina)

11 common mutations c.1521_1523delCTT p.(Phe508del) c.489+1G>T c.1624G>T p.(Gly542*) c.1652G>A p.(Gly551Asp) c.3846G>A p.(Trp1282*) c.1657C>T p.(Arg553*) c.1519_1521delATC p.(Ile507del) c.1679G>C p.(Arg560Thr) c.1646G>A p.(Ser549Asn) c.1647T>G p.(Ser549Arg) c.3909C>G p.(Asn1303Lys)

NIPD for cystic fibrosis: NGS for paternal exclusion

Hill et al. Prenatal Diagnosis 2015 epub March

Invasive Prenatal Diagnosis required if paternal mutation inherited

NIPD for Cystic fibrosis UKGTN approval 2014.

Congenital Adrenal Hyperplasia (CAH) – approaches to NIPD

• Sex determination

– Invasive testing is reduced by up to 50%

• Paternal mutation exclusion

– Complicated by highly homologous pseudogene and large deletions

– Not possible to test for specific mutations

– Linkage analysis to identify high risk paternal allele

– Further 25% reduction in invasive tests

• Relative haplotype dosage analysis (RHDO)

– Establish which maternal allele the fetus has inherited

– Requires accurate quantification of fetal fraction in cell free DNA

– Technically challenging – maternal background

Hap 1 / Hap 2

Maternal Paternal

Parental genotypes

Paternal Hap 1 transmitted if ‘C’ allele NOT detected

Or

A A A C

Fetal genotypes A A A C

Maternal plasma

Paternal Hap 2 transmitted if ‘C’ allele IS detected

Paternal haplotype transmission

NIPD to exclude maternal mutation?

Bespoke NIPD

• Around 1000 single gene disorder cases in RAPID Biobank, at risk of > 100 different conditions.

• Develop ability to offer NIPD to more high risk families. • Applicable where a mutation has been identified by invasive

prenatal or postnatal testing. Potential to offer testing in subsequent pregnancies

• Bespoke Assay design to detect paternal or apparently de novo mutation.

• Advance work-up

Added to panel:

Crouzon syndrome (3)

Cystic Fibrosis (5)

Family Specific:

Torsion Dystonia (4)

Fraser syndrome (3)

Osteogenesis imperfecta (3)

ARPKD (1)

*Tuberous sclerosis, NF1, SMARD1,

Zellweger, Rhabdoid Tumour

NIPD clinical service delivery at GOSH

In 2014 32% of our molecular PND for monogenic disorders were by NIPD

There is a trend towards increased uptake which may have economic impact

Gender ACH TD

Bespoke / Rare

NIPD Invasive NIPD Invasive NIPD NIPD

2008-9 103 21 4

2009-10 118 28 16

2010-11 103 27 13 21

2011-12 124 28 14 25 2

2012-13 163 20 22 17 11

2013-14 169 22 14 17 22 4

2014-15 158 25 28 26 22 12

2015-16 M1/2 23 4 11 3 5 7

Quality assurance

Plasmas extracted and analysed in duplicate and must be concordant

Maternal sample is run in parallel

Presence of fetal DNA determined using Y-specific sequences in male fetuses

HLA-B, HLA-A are used to detect the presence of the paternal allele as a control to confirm the presence of fetal DNA in female fetuses.

Reporting Policy Mutation positive - report Mutation negative, presence of cffDNA confirmed – report Mutation negative, can’t confirm presence of fetal DNA - retest

Implementing NIPD – laboratory considerations

Diagnostic NIPD is possible and available, particularly for paternal or de novo mutations.

How should we develop the NIPD service?

Health economics

High cost, low volume tests, no commercial interest

Paternal exclusion testing only cost neutral if samples can be multiplexed (TATS?)

NIPD for recessive conditions will be more expensive with SNP Haplotyping / RHDO

Clinical Utility - Who do we test?

We currently only offer NIPD for conditions where invasive PND is available

Should we be testing for information only?

Equity of access to safer testing for families at very high risk

UKGTN Approval process as NIPD becomes standard practice?

NIPD- Summary

Sequenom are consolidating their laboratories……………In addition, as part of

the consolidation, the firm said it would discontinue its SensiGene Fetal RHD

test.

The company decided to discontinue the SensiGene Fetal RHD test, which it

began offering in 2010, because it has not contributed substantially to its

revenues.

Genome Web 28.5.2015

2011 – UK Media Attention

By the end of 2014 >1,000,000 tests done worldwide

Offered in >90 countries

Mainly in the private sector with several countries researching implementation in public sector

Cost $350 - $2900

Profound impact on invasive testing

Non-Invasive Prenatal Testing

Chandrasekharan et al Sci Trans Med 2014; Warsof et al PND 2015

Advanced screening by non-invasive test to detect fetal aneuploidy

CB

T

NIP

T

UK NHS Trends in invasive tests

cffDNA is placental in origin – confined placental mosaicism

Fetal and maternal cfDNA is tested - unintended maternal diagnosis Maternal sex chromosome anomaly Maternal mosaicism Maternal tumours - abnormal cfDNA secreted from benign /malignant tumour Maternal transplants

‘Vanishing twin’

Other factors affecting performance Gestation High BMI Inconclusive results Failed results Variable performance between providers, no result <1-6%

NIPT for aneuploidy is not 100% accurate

False Positive Rate (%)

De

tectio

n R

ate

(%

)

0 10 20 30

70

75

80

85

90

95

10

0

1 in 150

1 in 1,000

1 in 2,0001 in 3,000

How could we offer NIPT for aneuploidy in the NHS? To all women undergoing DSS

NIPT 99% detection High false positive Decrease invasive testing Cost??? What do we do with failed NIPT Assume 75% uptake and a cost of £100 = £45 million

How could we offer NIPT for aneuploidy? Contingent Test

False Positive Rate (%)

De

tectio

n R

ate

(%

)

0 10 20 30

70

75

80

85

90

95

10

0

1 in 150

1 in 1,000

1 in 2,0001 in 3,000

>1:150 High Risk 85% detection for 3% false positive NIPT Decrease T21 detection slightly Decrease invasive testing

>1:1000 94-95% detection 12% False +ve NIPT Decrease invasive testing Affordable if NIPT <£250 £13.5 Million

To evaluate non-invasive prenatal testing (NIPT) for

aneuploidy in a NHS setting to provide information

for the National Screening Committee (NSC) that will

be used to inform decisions on introduction of NIPT

into the national Down Syndrome Screening (DSS)

programme.

NIPT for aneuploidy

EVALUATION

STUDY

Eight maternity units in London, SE England and Scotland

Offer NIPT to all women with a risk of >1:1000

IPD offered to all with DSS risk > 1:150 & to +ve

NIPT

Outputs

Evaluate DSS/NIPT/IPD uptake in real life

Evaluate test performance in an NHS service laboratory

Validated health professional education

Validated patient educational material

Service delivery plans

Detailed health economic evaluation

Formal report to the NSC

Evaluation of NIPT in the UK NHS

1st Nov 2013 – 28th Feb 2015

• Blood samples must reach lab within 24 hours

• Delay = ↑ contamination of cfDNA with mat DNA

• EDTA / Streck

– Fetal load appears to be consistent up to ~24 hours

– If transit time >24hrs, Streck should be used

• Spinning

– First spin 3,000rpm for 15mins

– Second spin 14,000rpm for 10mins

Blood draw and spinning 20mls from 11+ weeks gestation

cfDNA extraction on QIAsymphony 4mls maternal plasma

Library prep using TruSeq HT kit 50uL cfDNA

Whole Genome Sequencing

HiSeq 2500

Data Analysis

REPORT

Strategy overview

Laboratory Workflow

Blood draw and spinning 20mls from 11+ weeks gestation

cfDNA extraction on QIAsymphony 4mls maternal plasma

Library prep using TruSeq HT kit 50uL cfDNA

Whole Genome Sequencing

HiSeq 2500

Data Analysis

REPORT

Strategy overview

Laboratory Workflow

•Custom protocol for 4ml plasma, QIAsymphony DSP Virus/Pathogen Midi Kit •12 samples per reagent cartridge, •2.5hrs hands-off time •In house validation demonstrated efficient extraction compared with manual QIAgen cell free DNA extraction.

Blood draw and spinning 20mls from 11+ weeks gestation

cfDNA extraction on QIAsymphony 4mls maternal plasma

Library prep using TruSeq HT kit 50uL cfDNA

Whole Genome Sequencing

HiSeq 2500

Data Analysis

REPORT

Strategy overview

Laboratory Workflow

• 1 day

• Thorough internal validation required

• ~300 sample optimisation prior to pre-evaluation study

• >700 samples pre-evaluation study

Accurate quantification ESSENTIAL

• Bioanalyser & Qubit to quantify each individual library

• Pooled library re-quantified

Automated library clean-up •Qiacube •MinElute PCR Purification Kit •12 samples •30 minutes

Blood draw and spinning 20mls from 11+ weeks gestation

cfDNA extraction on QIAsymphony 4mls maternal plasma

Library prep using TruSeq HT kit 50uL cfDNA

Whole Genome Sequencing

HiSeq 2500

Data Analysis

REPORT

Strategy overview

Laboratory Workflow

•HiSeq 2500 •50bp from one end of each plasma DNA fragment sequenced •Rapid Run mode – 8 hours •24 samples per flow-cell, 16-plex >20million 30-70bp reads

Blood draw and spinning 20mls from 11+ weeks gestation

cfDNA extraction on QIAsymphony 4mls maternal plasma

Library prep using TruSeq HT kit 50uL cfDNA

Whole Genome Sequencing

HiSeq 2500

Data Analysis

REPORT

Strategy overview

Laboratory Workflow

1. Z-score – Mean and Standard Deviation

2. MAD-score – Median and absolute

deviation

3. Normalised Chromosome Value (NCV) – Within sample control

4. Principle component analysis (PCA) – GC correction

– Optimal removal of between sample variance

Reliant on large, consistent set of reference samples

Analysis tool – RAPID-R Lo et al Bioinformatics 2014

Method Masking Trisomy 21 Trisomy 18 Trisomy 13

sens spec sens spec sens spec

Z Y 97 100 89.5 99.9 14.3 100

Z N 95 100 87.2 99.9 28.6 99.7

MAD Y 98 100 89.5 99.9 21.4 99.9

MAD N 93 100 92.3 99.6 50.0 99.6

NCV Y 97.4 99.7 100 99.9

NCV N 97.4 99.9 100 99.7

PCA Y 98 98 97.4 99.9 100 99.7

PCA N 98 98 97.4 99.7 100 100

Bioinformatics

http://cran.r-project.org/web/packages/RAPIDR/index.html

Sens (%) Spec (%) CI (sens) CI (spec)

T21 100 100 87.54-100 98.07-100

T18 95.45 98 78.20-99.19 94.97-99.22

T13 100 99.53 72.25-100 97.38-99.92

XO 90.9 99.5 62.26–98.38 97.36–99.92

>1:150 6% declined all tests 77% NIPT 18% opted for IPD +/- NIPT

Didn’t understand DSS, would do nothing, 2 Harmony Mainly with scan abs, big NT or risk >1:10

1:151 – 1:1000

17% declined 83% NIPT

Had Harmony, decided against after thinking about it, content with risk 12 trisomy

NIPT evaluation study – Uptake

• 59 positive NIPT in 2555 samples

• 47 had confirmatory IPD

• 2 miscarried before IPD

• 10 declined IPD

• 5 discordant positive (1 T21, 4 T18)

• 33% elected to continue the pregnancy

• Of the women who opted for IPD directly 52 had an abnormal

result

• 7% continued the pregnancy

NIPT evaluation study – Results

Risk DS detected Invasive tests Miscarriages Cost

1:150 855 5721 29 £17 million

NIPT as a contingent test

1:1000 h 20% i 83.9% 5 + £7 million

1:500 h 18% i 84.3% 4 + 3.3 million

1:150 h 12% i 85.1% 4 -£330,000

Benefits and costs on a UK national level

Women very positive about NIPT with high uptake rate in both groups

i Invasive tests performed

h Detection of trisomies in high risk and intermediate group

Several women used NIPT to plan and prepare for the birth of a DS child.

Health Economic analysis indicates NIPT is cost neutral when testing high risk group

Conclusions

June 18th 2015 – Positive NSC response to the RAPID report. Consultation on implementation for high risk women anticipated.

NIPT can detect unbalanced rearrangements of >5Mb using standard sequencing

strategies and a variety of bioinformatics

NATERA

Targeted approach.

1p36-, 22q11.2-, 5p-, Angelman and Prader-Willi

Sequenom, Illumina, BGI

Whole genome MPSS targeted at selected regions

1p36-, 22q11.2-, 5p-, 4p-, Angeleman and Prader-Willi, Trisomies 16 and 22, 11q-, 8q- ……………

Screening for deletions with frequencies as low 1:100,000

Little published data

Largely based on spiked samples

More evidence required on sensitivity, specificity, positive predictive value

A non-targeted, whole genome approach is required for reasonable detection rates

Extending NIPT………..

NIPT delivers a highly sensitive and specific screening test for aneuploidy

Implementation into public sector maternity care is being considered

Should we be testing for information only?

Do the benefits of using NIPT to screen for sex chromosome aneuploidy outweigh the potential harms? Should we be reporting fetal sex at 10-11 weeks?

Is it acceptable to promote NIPT as a potential screen for microdeletion syndromes and cancer?

NIPT for aneuploidy - Summary

This report presents independent research funded by the National Institute for Health Research (NIHR) under its Programme Grants for Applied Research Programme (RP-PG-0707-10107 – “RAPID”). The views expressed are those of the author and not necessarily those of the NHS, the NIHR or the Department of Health.

Acknowledgements

The people

RAPID Team

NE Thames Regional Genetics Laboratory

Thousands of patients and

>40 UK FMUs and genetics units

The funding

NIHR PGfAR – RAPID programme grant

GOSH-UCL NIHR Biomedical Research Centre

Great Ormond Street Childrens Charity

Transforming prenatal care