6/8/2012

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Cell Free Fetal DNA: The Next Best Test?

Mary E Norton, MDProfessor of Obstetrics and Gynecology

Stanford University

Disclosures

• Principal Investigator of ongoing clinical trial on cfDNA supported by commercial entity (Ariosa Diagnostics)

• No personal financial involvement in any of the cfDNA companies

Fetal Cells in Maternal Circulation

• Georg Schmorl• German pathologist• Identified fetal trophoblasts in maternal lungs of women who died from eclampsia

Schmorl G, 1893

.

History of Non-Invasive Prenatal Testing� 1893: Schmorl� Long hiatus (nearly 100 yrs)� 1980’s: Development of cell sorting

◦ Allowed separation of single cellsDevelopment of PCR and FISH

◦ Allowed analysis of single cells� 1990’s: Great enthusiasm for NIPDx� 1997: Lo and others report on cell free fetal DNA� 2002: NIFTY trial� 2011: Introduction of noninvasive prenatal

diagnostic tests

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Noninvasive prenatal testing using intact fetal cells

• Intact (nucleated) cells carry entire genome with potential to provide DNA results or entire karyotype

• Different cell types have been studied, each have pros/cons

National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY)

Evaluated 2744 patients, 1292 w/male fetuses• 41% of cases with males were detected• 11% false positives• 74% detection rate for aneuploidy• 0.6-4% estimated FP rate

Bianchi et al, 2002

Intact Fetal Cells: Is There a Future?• Still appeal to this strategy• Entire fetal genome within each cell

Isolate intact fetal cell

Whole genome amplification

Microarray CGH

Cell free fetal DNA� In 1997, Lo et al reported on presence of fetal DNA in serum of pregnant women

� Y sequences found in DNA of 24/30 women carrying male fetuses and 0/13 women carrying female fetuses

� This was possible using a very small serum sample

� Subsequently confirmed by other investigators

Lo et al, Lancet, 1997

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Cell free fetal DNA

• Cell free fetal DNA (cffDNA) is made up of short segments of fetal nucleic acids that circulate in maternal plasma

• Origin of these fragments is thought to be primarily placenta

Cell free DNA results from apoptosis

Fetal DNA in Maternal Plasma: Characteristics• cffDNA represents ~10% of total DNA in maternal plasma (Lo 1998, Chiu 2011)

• Much higher percentage than intact fetal cells• cffDNAmade up of short (<200 bp) DNA fragments (Chan 2004)

• Reliably detected after 7 wks gestation (Birch 2005)• Higher concentrations late in gestation• Short half life (16 min), undetectable by 2 hrs postpartum (Lo 1999)

cffDNA: Clinical Applications• Gender determination

• Single gene disorders: ▫ Detect paternally inherited allele

• Isoimmunization: noninvasively determine fetal Rh type

�Aneuploidy: detect abnormal ratio of a particular chromosome

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Aneuploidy Testing• Largest area of commercial interest• 4+ million births/year x NEW TEST = $$$

Companies currently offering cfDNAtesting:

Noninvasive Prenatal Testing

• Detection requires accurate quantification of DNA from a specific chromosome

• Several different methods are being utilized

cffDNA: Clinical ChallengesDistinguishing fetal DNA still challenging:• Concentration of all cell free DNA is low• Total amount varies between individuals• Fetal DNA molecules are outnumbered 20:1 by maternal cell free DNA molecules�Requires strategy to overcome preponderance of maternal DNA

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Importance of Fetal DNA Fraction• Trisomy detection via cfDNA depends on fraction of

DNA that is fetal• The higher the fetal fraction, the easier it is to detect

trisomy

Trisomy 21Disomic Chr

Total: 100(Maternal: 90)

10% fetal DNA in circulation

Total: 105(Maternal: 90)

20% fetal DNA in circulation

Total: 100(Maternal: 80)

Trisomy 21

Total: 110(Maternal: 80)

Disomic Chr

cffDNA: Clinical ChallengesFalse negatives: • Failure to extract adequate material• Individual variation in amount of cffDNAFalse positives:• Contamination• Unrecognized or vanishing twin• Placental mosaicism• Low level maternal mosaicism

Next generation sequencingBig advance allowing cfDNA testing has been development of next generation sequencing

• Initial DNA sequencing entailed radiation-based methods; manually loading electrophoresis gels and reading bases from the resulting images

• Next generation sequencing is automated, and therefore easier, faster and cheaper

• There are many platforms and methods but overall theme is rapidly improving methods and decrease in costs

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Next Generation Sequencing

http://gcat.davidson.edu/

Massively Parallel Sequencing

• Many cffDNA tests use this approach

• “Massive”: tons of DNA sequencing data

• “Parallel”: many pieces of DNA sequenced at the same time

Analysis of fetal DNA

Zhong, X, Holzgreve, W, Glob. libr. women's med 2009

Massively Parallel Shotgun Sequencing • MPSS is a random sampling of cfDNA fragments from all chromosomes• A z-score value is used as a cut-off for non-trisomy (Sequenom

Materni21)

Palomaki GE et al. (2011), Genet. Med

N=1696

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MPSS using cffDNA (Materni21)• n= 4664 high risk women (CVS/amnio)• 212 with trisomy 21; 1484 controls▫ 1% uninterpretable: QNS, poor fetal fraction; higher with high BMI

• DR: 98.6% (209/212)• FP rate: 0.2% (3/1471)• Excluded twins, IVF; 89% Caucasian�Clearly far better than current screening, “near diagnostic” but very expensive

Palomaki et al. Genet Med, October 2011

Massively Parallel Shotgun Sequencing

• “No call” zone for values between 2.5-4

• Disproportionate number of positives in this zone

N=532Bianchi DW et al. (2012) Obstet Gynecol

MPSS using cffDNA (Verifi)

• N=2882 high risk women (CVS/amnio)• N=532 analyzed▫ N=221 abnormal karyotypes

• DR 100% for T21 (89/89)• FPR 0% (none in 404 normal cases)• However, cases with a midrange risk were considered “unclassified” ▫ several had aneuploidy

Bianchi DW et al. (2012) Obstet Gynecol

Selective Sequencing and Risk Calculation (Harmony)Another approach involves selective sequencing of chromosomes 18 and 21, and risk calculation based on:

• cfDNA counts• Fraction of fetal DNA• Maternal and gestational age

More like serum screening results

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T21 Detection

Selective sequencing approach

� Multicenter cohort study of 3228 patients� Trisomy 21� 81/81 cases detected (100%)� 1/2888 false positive for T21 (0.03%)

� No result obtained (test failure) in 4.6%� Low fraction of fetal DNA� Failed sequencing

Norton et al, AJOG, 2012

MPSS for Trisomy 21 -- Detection Rate and False Positive Rate

Author DR FPR

Palomaki ’11 99% 0.1%Bianchi ’12 100% 1.5%Norton ’12 100% 0.03%

CA State Screen 90% 5%

cffDNA for Aneuploidy – T13 and T18

Author T13 T18 T21DR FPR DR FPR DR FPR

Palomaki ’11 92% 0.5% 100% 0.7% 99% 0.1%Bianchi ’12 81% 0 97% 0.6% 100% 1.5%Norton ’12 n/a n/a 97.4% 0.07% 100% 0.03%

CA State n/a n/a 81% 0.31% 90% 5%

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Fetal fraction of DNA and test failure

• 2-5% of samples do not provide a result

▫ Low fraction fetal DNA, failed sequencing, high variability in counts

▫ Some association with gestational age (<10 wks) and maternal BMI

Only validated in high risk patients• Positive results need to be taken in the context of disease prevalence• The less prevalent a disease, the more likely a positive test is a false positive

Test accuracy:

99% detection

0.2% false positive

T21 prevalence:

1 in 1,000

1,000 women

1 T21 999 non-T21

1 2 9970

Test + Test +Test - Test -

Positive test result is truly right only 1/3 of the time at extreme test performance

Why Low False Positive Rate Matters

# of true trisomies

# of false positives at different FPR5% 1% 0.5% 0.1%

T21(1 in 740) 7 250 50 25 5

T18(1 in

5,000)1 250 50 25 5

T13(1 in

16,000)0 250 50 25 5

Example: Ob/Gyn practice with 5,000 births/yr

The less common a genetic condition, the more important it is to have a low false

positive rate

Aneuploidy Screening

• Clinical tests have been introduced• However, at present they are not 100% accurate▫ Twin demise, mosaicism

• Patients should not terminate based on a maternal blood test▫ Invasive confirmation will still be necessary�Better screening test (vs truly diagnostic)

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cffDNA: Where Does it Fit?

10 11 12 13 14 15 16 17 18 19 20

Weeks of Gestation

First Trimester Serum Second Trimester Serum

NT Ultrasound

CVS Amnio

cffDNA: Where Does it Fit?

10 11 12 13 14 15 16 17 18 19 20

Weeks of Gestation

First Trimester Serum Second Trimester Serum

NT Ultrasound

CVS Amnio

cffDNA: Where Does it Fit?

10 11 12 13 14 15 16 17 18 19 20

Weeks of Gestation

NT Ultrasound?

CVS Amnio

cffDNA: Where Does it Fit?

10 11 12 13 14 15 16 17 18 19 20

Weeks of Gestation

First Trimester Serum Second Trimester Serum

NT Ultrasound

CVS Amnio

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cffDNA: Where Does it Fit?

10 11 12 13 14 15 16 17 18 19 20

Weeks of Gestation

First Trimester Serum Second Trimester Serum

NT Ultrasound

Amnio

Amnio

Aneuploidy Testing Timeline

Screening

Results

Diagnostic testing

Results

Decisions

Current Screening Timeline

Aneuploidy Testing Timeline

Screening

Results

Diagnostic testing

Results

Decisions

Screening

Results

cffDNA

Results

Diagnostic testing

Results

Decisions

Current Screening Timeline cfDNA as secondary screen

Where does cffDNA fit?

�Is this an outstanding screening test or an imperfect diagnostic test?

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Excellent first line screening test

Will simplify the process

• Now: 2 blood tests and an US▫ DS risks given twice▫ Narrow GA window

• cffDNA: Single blood test at any GA

Aneuploidy Testing vs Current Screening Algorithms

Pros� Simpler protocol◦ Results more straightforward

◦ Not as gestational age dependent

� More accurate◦ Fewer invasive tests

� Potentially earlier results

Cons� Need for more complete

consent◦ Advantages of a two-step process

� Fewer invasive tests = less expertise

� 2-4% test failure� Expensive

American College of Obstetricians and Gynecologists Opinion:

American College of Obstetricians and Gynecologists Opinion:

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Other Professional Societies• International Society for Prenatal Diagnosis• National Society of Genetic CounselorsThere are recognized benefits, but…▫ Not diagnostic� Needs confirmation� “Advanced screening test”

▫ T18 and T21 are not only aneuploidies detected by invasive testing

▫ Requires comprehensive genetic counseling▫ Need a low risk study before introducing into general population screening

What is the Future of:• Serum screening?▫ 1st trimester▫ 2nd trimester

• Amniocentesis?• CVS?• Cell free fetal DNA?• Microarray based Comparative Genomic Hybridization (aCGH)?

• Nuchal Translucency Screening?

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Summary• cfDNA is here to stay for aneuploidy testing• Exact role is still being clarified• This talk was completed 5/10/12 at 4:45 pm (PST) was out of date yesterday▫ New papers published and tests available this week

• Sequencing of the fetal genome is likely to be a reality in the not-too-distant future▫ Reported yesterday (6/5/12) in Science

• Tremendous clinical and ethical issues and considerations