SCREENING FOR FETAL ANEUPLOIDIES USING MATERNAL BLOOD: INTACT - PowerPoint PPT Presentation

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 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

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)

GENERAL STRATEGY (1990s, early 2000s) FOR RECOVERY OF INTACT FETAL CELLS Centrifugation PCR Recover 1 to 4 fetal Mononuclear Separation cells per ml Cell Layer (Ficol; 1.077 gm/ml) FISH Enrichment 1 in 10 3 MACS FACS to 10 4 (CD71 + /Gamma Globin + )

FIVE-COLOR FISH TO DETECT FETAL TRISOMIC CELLS IN ENRICHED POPULATION FROM MATERNAL BLOOD Trisomy 18 Trisomy 21 Bischoff et al., Am J Obstet Gynecol 1998

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

NEW APPROACHES FOR INTACT FETAL CELLS (2004 - ) • Automated microscopy and FISH to analyze any rare cells present. • New generation flow cytometry or magnetic activated sorting. • MEMS and other devices to capture cells.

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

MICROFLUIDICS Mathematically -modeled flow rate and post placement to maximize cell capture

FirstCEE TM 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

FETAL CELL TYPES • Nucleated red blood cells (Maternal blood) • Trophoblasts (Maternal cervical mucus; maternal blood)

CONFIRMATION OF CAPTURED TROPHOBLASTS BY POSITIVE STAINING INSIDE CHANNELS Trophoblast staining

TROPHOBLASTS • Facile analysis of cells not achieved (FISH) (Bischoff and Simpson, Biocept, 2006) 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

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

Single cell laser microdissection ISET isolated cell STR (Short Tandem Repeats)/ genotyping (CA)1; (CA)3 CACACA Father’s DNA CA (CA)5; (CA)7 CACACACACACACA Mother’s DNA CACACACACA (CA)1; (CA)7 CACACACACACACA Fetal cell DNA CA 10 genomic analysis on the genome of a single cell Vona et al, Am J. Pathol, 2002

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

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)

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.

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

RhD and RhCc/Ee Locus 99 bp 113 bp D CcEe Rh(D) d CcEe Rh(d)

Fluorescent PCR detection of RhD and RhCc/Ee

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.

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.

TARGETING SEQUENCES • Determine total chromosome 21 transcripts maternal and fetal • Trisomic pregnancies should be 2.5% greater than normal pregnancies

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

Fan H. C. et.al. PNAS 2008;105:16266-16271

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

CELL-FREE DNA Digital PCR- Template- Quantification Lo YMD, Lun FMF, Chan KCA et al, PNAS, 2007.

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”

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)

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)

Chiu et al., 2011

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

Fig 5 Ehrich et al.,2011

Fig 5 Ehrich et al.,2011

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

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.

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.