Transcript Slide 1

Analysis of free fetal DNA in maternal blood:
Options currently available for screening use
Jack Canick
Intensive Course on Antenatal Screening for Down’s Syndrome
Wolfson Institute of Preventive Medicine
London
May 2013
Outline
• Commerical tests currently available (their DR, FPR,
no call claims;chromosomes tested)
• Intellectual property associated with DNA testing
• Professional society statements/guidelines
• Primary screening as usual with usual cut-offs
• Secondary screening with DNA
• Two step reflex screening (DNA the secondary test)
• Use DNA as the primary screening test (drop current
screening)
Maternal Plasma DNA Testing in the U.S.
Sequenom Center for Molecular Medicine
San Diego, California
Verinata Health
Redwood City, California
Ariosa Diagnostics
San Jose, California
Natera Inc.
San Carlos, California
Maternal Plasma DNA Testing Outside the U.S.
Lifecodexx AG
Konstanz, Germany
PrenaTest®
Beijing Genomics Institute
Shenzhen, China
NIFTY Test
Berry Genomics
Beijing, China
BambniTest
• massively parallel sequencing
• method licensed from Sequenom
• marketing with and without partners
in Europe, Turkey, Dubai
• massively parallel sequencing
• marketing in China, Asia-Oceania,
Europe, Middle East ...
• have new labs in Copenhagen, U.S…
• massively parallel sequencing
• marketing in China, and with partners
in Asia-Oceania, Israel…
• Collaboration with Baylor College of
Medicine?
Performance of Currently Available DNA Tests
Test performance criteria:
Detection Rate (DR)
proportion of affecteds
called positive by the test
False Positive Rate (FPR) proportion of unaffecteds
called positive by the test
No-Call Rate
proportion of tests that
are not reported
Performance of Currently Available DNA Tests
No-Call Rate: reasons
fetal fraction too low
<3% FF or <4% FF
fetal fraction too high
>50% FF
testing failures
multiple steps possible
choice of rules/cut-offs
program decisions
results cannot be explained
biology/sample preparation?
sample handling errors
sample in transit/lab
No-Call Rate: expectations
from publications
1-6% - method dependent
Performance of Currently Available DNA Tests:
Focus on Screening for Down Syndrome
Performance of Currently Available DNA Tests:
Most recent published results on Trisomy 21
Publication from academic source or vendor
Sequenom
20111
Verinata
20122
Ariosa
20123
Natera
20134
BGI
20125
DR
98.6%
100%
100%
100%
100%(>95%)
FPR
0.2%
0%
0.03%
0%
0.01%
No-Call
Rate
0.8%
4.3%
4.6%
5.4%
0.7%
1. Palomaki GE et al. Genet Med 2011;13:913-20.
2. Bianchi DW et al. Obstet Gynecol 2012;119:890-901.
3. Norton ME et al. Am J Obstet Gynecol 2012;207:107.e1-8.
4. Nicolaides KH et al. Prenat Diagn 2013;33 on-line
5. Dan S et al. Prenat Diagn 2012;32:1-8.
DR = 209/212 = 98.6%
FPR = 3/1471 = 0.2%
13 no-calls
13/1696 = 0.8%
DR = 89/89 = 100%
FPR = 0/404 = 0%
23 no-calls
23/532 = 4.3%
DR = 81/81 = 100%
FPR = 1/2888 = 0.03%
148 no-calls
148/3228= 4.6%
Prenatal DiagnosisEarly View,
Article first published online: 24 APR 2013
DR = 25/25 = 100%
FPR = 0/204 = 0%
13 no-calls
13/242= 5.4%
Maternal blood samples (n= 11,184)
Sample failed QC for sequencing (DNA extraction, library
prep, or sequencing) (n=79)
Pregnant women received the MPS-based test (n=11,105)
DR = 140/140 = 100%*
FPR = 1/11,105 = 0.01%
Test negative (n=10,915)
Validated by
karyotyping,
non-T21 and nonT18 (n=2,818)
Follow-up investigation
(n=8,097:
Normal (n=4,524)
Pregnany termination
(n=70)
Stillbirth (n=15)
Others (n=13)
unknown (n=32)
Lose contact (n=3,503)
Test positive (n=190)
Validated by
karyotyping
(n=182):
T21 (n=140; 1 FP)
T18 (n=42; 1 FP)
Follow-up
investigation (n=8):
Induced abortion
(n=5)
Stillbirth (n=2)
Spontaneous
abortion (n=1)
79 no-calls
79/11,184= 0.7%
* 8 screen positives
with unknown
karyotypes
DR >140/148 = >95%
Performance of Currently Available DNA Tests:
Aneuploidies other than Down syndrome
Performance of Currently Available DNA Tests:
Trisomy 18 and Trisomy 13
Publication from academic source or vendor
Screening Performance (DR and FPR)
Sequenom Verinata
20121
20122
Ariosa
20123
Natera
20134
BGI
20125
ALL STUDIES
42/42
1/11105
99% (176/178)
0.08% (13/16665)
---
89% (25/28)
0.06% (15/2696)
trisomy 18
DR
FPR
59/59
10/1971
35/36
0/497
trisomy 13
DR
FPR
13/13
15/1971
11/14
0/521
37/38
3/3
2/2888 0/204
---
1/1
0/204
1. Palomaki GE et al. Genet Med 2012;14:296-305.
2. Bianchi DW et al. Obstet Gynecol 2012;119:890-901.
3. Norton ME et al. Am J Obstet Gynecol 2012;207:107.e1-8.
4. Nicolaides KH et al. Prenat Diagn 2013;33 on-line
5. Dan S et al. Prenat Diagn 2012;32:1-8.
presentation by Charles Strom, MD, of Quest Diagnostics
http://education.questdiagnostics.com/presentations/noninvasive-prenatal-testing-separate-but-not-equal?presentation_id=235
Intellectual Property: Lo et al.
The ‘540 Patent
Licensed to Sequenom; Patent ends: Nov 29, 2019
Intellectual Property: Quake et al
Massively Parallel Sequencing
Licensed to Verinata; Patent ends February 26, 2029
Intellectual Property: Fan & Quake
Diagnosis by Sequencing (The ‘076 Patent)
Licensed to Verinata; Patent ends April 12, 2032
Intellectual Property: Lo et al
Massively Parallel Sequencing (patent pending)
Intellectual Property: Law Suits
From: BioPharm Insight, Feb 12, 2012:
• Sequenom is engaged in litigation with… Aria Diagnostics and Natera.
• Verinata Health announced on 23 Feb [2011] that it has commenced an action
against Sequenom, seeking a declaratory judgment that its [methods] do not
infringe the ‘540 patent.
• Aria filed suit against Sequenom in Dec [2012] claiming that Sequenom is
overly aggressive in its enforcement of the ‘540 patent.
• Sequenom then filed against Aria in Jan [2012] alleging Aria infringes the ’540
patent, and the infringement has been “intentional, deliberate, and willful.”
• Natera similarly filed suit against Sequenom in January [2012], claiming it
does not infringe the ‘540 patent and that the patent is invalid.
• Sequenom subsequently filed suit [ against Natera] claiming infringement
From the Verinata Health website:
• Verinata files suit against Ariosa and LabCorp claiming infringement of their
‘076 patent.
Professional Society Statements/Guidelines
“Maternal cfDNA screening is an emerging
technology that can provide highly effective
prenatal screening for Down syndrome, trisomy
18, and possibly trisomy 13 in high risk women.
It is not a replacement for the analysis of
amniotic fluid cells or CVS.”
ACOG Committee Opinion No. 545, Dec 2012
• …offers tremendous potential as a screening tool for
fetal aneuploidy..
• …cell-free fetal DNA can be used as a…screening test in
women at increased risk of aneuploidy.
• A patient with a positive test result should be referred for
genetic counseling and should be offered invasive
prenatal diagnosis for confirmation of test results.
• Cell free fetal DNA testing should not be offered to lowrisk women or women with multiple gestations because it
has not been sufficiently evaluated in these groups.
ACMG Statement, February 2013
Noninvasive prenatal screening
for fetal aneuploidy
• Non-invasive [fetal DNA] testing offers tremendous
potential as a screening tool…
• …should not be offered to low-risk women or women
with multiple gestations because it hasn’t been
sufficiently evaluated in these groups.
• A patient with a positive test result... Should be offered
invasive prenatal diagnosis…
Other Professional Society Statements/Guidelines
2013
Canada
SOGC
2013
Australia/New Zealand
RANZCOG
I was not able to find statements or
guidelines from other countries.
How best to implement
maternal plasma DNA testing?
How best to implement maternal plasma DNA testing
As a secondary screening test (2 steps)
1
Conventional test
Screen negative
(no further action)
Result reported; patient
Screen positive must decide on DNA test
or invasive test
2
DNA test
Screen negative
(no further action)
Screen positive
Invasive diagnostic test
(CVS/amnio)
How best to implement maternal plasma DNA testing
As a reflex screening test (1 step)
1
Conventional test
+ maternal plasma hold tube
Screen negative
(no further action)
Interim positive
Use more generous FPR.
Result not reported.
All screen positives
reflexed to DNA test
DNA test
Screen negative
(no further action)
Screen positive
Invasive diagnostic test
(CVS/amnio)
How best to implement maternal plasma DNA testing
As a primary screening test (1 step)
1
Maternal plasma DNA test
Screen negative
(no further action)
Screen positive
Invasive diagnostic test
(CVS/amnio)
Performance of conventional screening
tests with and without DNA-based
secondary screening.
Example: Consider 100,000 women from the
general pregnant population, with a Down
syndrome prevalence of 1:500:
200 will have Down syndrome
99,800 will not have Down syndrome
Screening:
1st Trimester
Combined test
alone
screen
positives
are offered
invasive
testing
100,000 pregnant women
(Down syndrome prevalence: 1 in 500)
1st trim combined test
85% DR 5% FPR
DS identified
false positives
170
OAPR: 1:29 (3.3%)
4,990
PPV
Screening:
1st Trimester
Combined test
followed by
DNA test
100,000 pregnant women
(Down syndrome prevalence: 1 in 500)
1st trim combined test
85% DR 5% FPR
DS identified
false positives
screen positives
(5%) are offered
DNA test
If still screen
positive,
offer
invasive
testing
170
OAPR: 1:29 (3.3%)
4,990
PPV
DNA test
99% DR 0.2% FPR
DS identified 168 of 200 = 84% DR
false positives 25 of 4,990 = 0.5% FPR
OAPR: 17:1 (94% PPV)
Screening:
1st Trimester
Combined test
with reflex to
DNA test
Do not report result
at this time
100,000 pregnant women
(Down syndrome prevalence: 1 in 500)
1st trim combined test
93% DR 10% FPR
If screen positive, do the DNA
test on the held sample
DNA test
99% DR 0.2% FPR
If still screen positive,
offer invasive testing
If still screen
positive,
offer
invasive
testing
DS identified
false positives
OAPR
184 of 200 = 92% DR
20 of 99,800 = 0.02% FPR
9:1 (90% PPV)
Screening:
Integrated test
alone
100,000 pregnant women
(Down syndrome prevalence: 1 in 500)
Integrated test
90% DR 2% FPR
screen
positives
are offered
invasive
testing
DS identified
false positives
180
OAPR: 1:11 (8.3%)
1,996
PPV
Screening:
Integrated test
followed by
DNA test
100,000 pregnant women
(Down syndrome prevalence: 1 in 500)
Integrated test
90% DR 2% FPR
DS identified
false positives
screen positives
(2%) are offered
DNA test
If still screen
positive,
offer
invasive
testing:
180
OAPR: 1:11 (8.3%)
1,996
PPV
DNA test
99% DR 0.2% FPR
DS identified 178 of 200 = 89% DR
false positives
4 of 1,996 = 0.5% FPR
OAPR: 44:1 (98% PPV)
Screening:
Integrated test
with reflex to
DNA test
100,000 pregnant women
(Down syndrome prevalence: 1 in 500)
Integrated test
98% DR 10% FPR
Do not report result
at this time
If screen positive, run the DNA
test on the held sample
DNA test
99% DR 0.2% FPR
If still screen positive,
offer invasive testing
If screen
positive,
offer
invasive
testing
DS identified
false positives
OAPR
194 of 200 = 97% DR
10 of 99,800 = 0.01% FPR
19:1 (95% PPV)
Screening:
DNA test
alone
100,000 pregnant women
(Down syndrome prevalence: 1 in 500)
DNA test
99% DR
0.2% FPR
If screen positive, offer invasive testing:
If screen
positive,
offer
invasive
testing:
DS identified
false positives
OAPR
198 (of 200 = 99% DR)
200 (of 99,800 = 0.2% FPR)
1:1 (50% PPV)
Finally, how to deal with No-Calls
When maternal plasma DNA is a secondary test:
• These no-calls are already considered high risk.
• Therefore, even without a DNA test result, they are
candidates for invasive diagnostic testing.
• So, they are considered part of the screen positive group,
and contribute to the false positive rate of the DNA test.
When maternal plasma DNA is a primary test:
• These no-calls can be tested using conventional
screening methods.
• Therefore, most no-calls will be screen negative.
• Those that are screen positive are candidates for invasive
diagnostic testing.