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SCREENING FOR FETAL ANEUPLOIDIES USING MATERNAL BLOOD: INTACT FETAL CELLS AND CELL FREE DNA JOE LEIGH SIMPSON, M.D. Florida International University Miami, Florida USA Orlando 2011 APPROACHES TO PRENATAL GENETIC DIAGNOSIS Invasive

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SCREENING FOR FETAL ANEUPLOIDIES USING MATERNAL BLOOD: INTACT FETAL CELLS AND CELL FREE DNA

JOE LEIGH SIMPSON, M.D. Florida International University Miami, Florida USA

Orlando 2011

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APPROACHES TO PRENATAL GENETIC DIAGNOSIS

  • Invasive procedure offered directly

(amniocentesis, CVS)

  • Noninvasive screening followed by

invasive procedure if fetal risk high; 1 in ~ 15-25 procedures will reveal abnormality

  • Definitive noninvasive diagnosis with

procedure only (rarely) to confirm; virtually all procedures should reveal abnormality

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CIRCULATING CELLS AND DNA IN BLOOD: PREGNANCY

  • First to detect fetal aneuploid cells in

maternal blood:

  • Trisomy 18

(Price, Elias, Wachtel, Simpson; 1991)

  • Trisomy 21

(Elias, Price, Doktor, Simpson; 1992)

  • 1994-2003 National Institutes of Health

Fetal Cell Study Group

(Bianchi, Bischoff, Elias, Evans, Holzgreve, Jackson, Lewis, Simpson)

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Separation (Ficol; 1.077 gm/ml)

Recover Mononuclear Cell Layer

Centrifugation

Enrichment

MACS FACS (CD71+/Gamma Globin+)

PCR 1 to 4 fetal cells per ml

GENERAL STRATEGY (1990s, early 2000s) FOR RECOVERY OF INTACT FETAL CELLS

FISH 1 in 103 to 104

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Trisomy 21 FIVE-COLOR FISH TO DETECT FETAL TRISOMIC CELLS IN ENRICHED POPULATION FROM MATERNAL BLOOD Trisomy 18

Bischoff et al., Am J Obstet Gynecol 1998

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CONCLUSIONS (NIH): INTACT FETAL ERYTHROBLASTS

FISH to Detect Aneuploidies:

  • 74% detection of fetal aneuploidy

analyzing slides by fluorescent in situ hybridization (FISH); MACS preferable to FACS

  • Enrichment and analysis inefficient

and not consistently achieved. NICHD recommended biotech collaboration

Bianchi, Simpson, Jackson

  • Prenat. Diag., 2002
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NEW APPROACHES FOR INTACT FETAL CELLS (2004 - )

  • Automated microscopy and

FISH to analyze any rare cells present.

  • New generation flow cytometry
  • r magnetic activated sorting.
  • MEMS and other devices to

capture cells.

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CEE combines attachment chemistry and fluid dynamics designed to isolate cells needed for highly accurate genetic test results

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Mathematically

  • modeled flow

rate and post placement to maximize cell capture

MICROFLUIDICS

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FirstCEETM VALIDATION – FETAL ERYTHROBLASTS IN MATERNAL BLOOD

  • Steady progress until cessation (Oct 2008)

due to company (Biocept) prioritization toward detection of cancer cells: – Aneuploidy (FISH) successfully detected in most pregnancies having male fetuses, as verified by PCR and Y-FISH signal – Little to no false positives in over 6,000 samples – Difficulties in distinguishing XX fetal from XX maternal cells, using epsilon as fetal marker

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FETAL CELL TYPES

  • Nucleated red blood cells

(Maternal blood)

  • Trophoblasts

(Maternal cervical mucus; maternal blood)

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CONFIRMATION OF CAPTURED TROPHOBLASTS BY POSITIVE STAINING INSIDE CHANNELS

Trophoblast staining

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TROPHOBLASTS

Explanations 1) Trophoblasts already degenerating. 2) Trophoblasts too fragile for analysis. Solutions (Paterlini-Bréchot) 1) Fixation conferring cellular robustness 2) Molecular analysis individual microdissecting cells

  • Facile analysis of cells

not achieved (FISH) (Bischoff and Simpson, Biocept, 2006)

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ISET (Isolation by size of Epithelial Tumor/Trophoblastic cells) Vona G et al, Am J Pathol, 2000

CTC CFTC

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Single cell laser microdissection

10 genomic analysis on the genome of a single cell

Father’s DNA Mother’s DNA Fetal cell DNA

CACACA CA CACACACACACACA CACACACACACACA CACACACACA CA (CA)1; (CA)3 (CA)5; (CA)7 (CA)1; (CA)7

STR (Short Tandem Repeats)/ genotyping

Vona et al, Am J. Pathol, 2002

ISET isolated cell

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CLINICAL UTILITY OF TROPHOBLASTS (Paterlini-Bréchot)

  • Proof of principle reports (SMA,

Lancet, 2003; cystic fibrosis,

  • Prenat. Diag., 2006)
  • Consecutive cases (cystic fibrosis

and SMA) successfully diagnosed

International Society Prenatal Diagnosis, 2010

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CELL FREE FETAL DNA IN MATERNAL BLOOD

  • Initially recovered from plasma by

Lo (1990s)

  • Now generally recovered from whole

blood

  • Size fractionation (50-200 bp fetal) or

differentially methylated genes (fetal > maternal)

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CELL FREE FETAL DNA TO DETECT PATERNAL ALLELE (THUS FETAL ALLELE) NOT PRESENT IN MOTHER

  • 1. Paternal mutations to detect mendelian

mutation being transmitted to fetus (e.g., Marfan, Huntington). Presence of mutant DNA in mother must be derived from affected fetus.

  • 2. Rh(D) to distinguish Rh negative (d/d)

from Rh(D/d) fetus given RhD/d father D in maternal blood can occur only if of fetal origin.

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CELL FREE FETAL DNA TO DETECT PATERNAL Rh(D)

  • Rh negative (d/d) mother at risk for

sensitization if fetus Rh positive (D/d)

  • Heterozygous Rh (D/d) father can

transmit either D or d to fetus

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RhD and RhCc/Ee Locus

CcEe D

Rh(D) Rh(d)

d CcEe

99 bp 113 bp

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Fluorescent PCR detection of RhD and RhCc/Ee

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CURRENT STATUS CELL FREE DNA for Rh(D)

  • Standard practice in many

European countries, but not yet standard in U.S.

  • Multiple U.S. vendors will offer

testing for fetal gender and Rh(D) as first application of single gene cell free DNA in maternal blood.

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CELL FREE FETAL DNA FOR ANEUPLOIDY DETECTION

  • Strategy: Increased trisomy 21

transcripts (maternal and fetal) in maternal blood of trisomic pregnancies compared to maternal blood of euploid (normal) pregnancies.

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TARGETING SEQUENCES

  • Determine total chromosome 21

transcripts maternal and fetal

  • Trisomic pregnancies should be

2.5% greater than normal pregnancies

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INCREASED TOTAL MATERNAL AND FETAL TRANSCRIPTS IN MATERNAL BLOOD IN TRISOMES

  • Nos. 21 DNA

Disomy Trisomy Total

Total No. 21 Fetus Fetus

  • Nos. 21 Transcripts

Mother 2 2 4 95 + 5 = 100* Fetus 2 3 5 95 + 7.5 = 102.5

  • Assume 5% of cell free DNA of fetal origin
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Fan H. C. et.al. PNAS 2008;105:16266-16271

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DIGITAL PCR

  • Populate wells with probes for

chromosome 21 DNA.

  • Expose wells to dilute DNA from

maternal blood, and count number of wells containing or overexpressing chromosome 21 - specific transcripts

  • Number of wells overexpressing 21

transcripts should be greater if trisomic fetus present

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CELL-FREE DNA Digital PCR- Template- Quantification

Lo YMD, Lun FMF, Chan KCA et al, PNAS, 2007.

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PREREQUISITE FOR CLINICAL INTRODUCTION OF CELL FREE OR INTACT FETAL CELLS FOR ANEUPLOIDY DETECTION

  • Ability to obtain a result consistently

(need not be 100%)

  • Accurate results, especially in

excluding fetal trisomy

  • Ability to process sufficient number of

samples to meet demand (automation?)

  • Ability to address clinical confounders,

e.g., “vanishing twin”

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CELL FREE FETAL DNA FOR TRISOMY 21 DETECTION

  • 753 pregnant women at high risk for

trisomy 21 (prospective obstetrics cases and archived plasma samples)

  • 1.7% failed recruitment criteria
  • 5.6% failed specimen quality criteria
  • 753 tested 8-plex (8 samples concurrently)
  • 314 tested 2-plex (2 samples concurrently)

Chiu et al. (Brit Med J. 2011;342:c7401)

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CELL FREE FETAL DNA FOR TRISOMY 21 DETECTION (CHIU ET AL., 2011)

Sensitivity Specificity 8 plex 79.1% 98.9% 2 plex 100% 97.9% Plex = # samples concurrently analyzed

Chiu et al. (Brit Med J. 2011;342:c7401)

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Chiu et al., 2011

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CELL FREE FETAL DNA FOR TRISOMY 21 DETECTION (EHRICH ET AL., 2011)

  • 480 archived samples
  • Massive parallel shotgun

sequencing of cell free fetal DNA using “…several process improvements”

Ehrich AJOG 2011;204:25.e1-11

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SLIDE 34

Fig 5

Ehrich et al.,2011

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Fig 5

Ehrich et al.,2011

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DEFINITIVE NONINVASIVE PRENATAL GENETIC DIAGNOSIS: STATUS in 2011

Single Gene

  • Clinically applicable (reliable) and little

limitation except pragmatism –for excluding RhD fetus –for excluding transmission any mutant paternal allele –For excluding de novo mutations, particularly given ultrasound anomaly

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DEFINITIVE NONINVASIVE PRENATAL GENETIC DIAGNOSIS: STATUS in 2011

  • 1. Cell free fetal DNA aneuploidy or

“screening” available soon, but will not be labeled “test”.

  • 2. Cell free DNA tests will be first to market

but in 3+ years intact fetal cell(s) will be available and provide much more information.

  • 3. Less than 100% informative as single test

but could be repeated 1-2 weeks later.

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DEFINITIVE NONINVASIVE PRENATAL GENETIC DIAGNOSIS: STATUS in 2011

  • 4. Accurate in excluding aneuploidy; false

positives will be rare but enough to require confirmation before termination.

  • 5. Could be available earlier (6-8 weeks

gestation) in pregnancy than invasive tests but may not be offered initially

  • 6. Expense likely an issue if no more than 2

samples can be tested concurrently.