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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
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