Biggio UAB Nursing Conference 2014
Growing Options in Prenatal Screening & Diagnosis
Joseph R. Biggio Jr., M.D. University of Alabama at Birmingham
Disclosures
• Site Investigator at UAB for trials enrolling patients • Natera Prenatus study • Sequenom • No personal support or other conflicts
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CLINICAL MANAGEMENT GUIDELINES FOR OBSTETRICIAN—GYNECOLOGISTS NUMBER 77, JANUARY 2007 Screening for Fetal Chromosomal Abnormalities
ACOG Practice Bulletin #77
• Maternal age should not be sole factor in offering diagnostic test
• Diagnostic testing should be an option for all women
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ACOG Practice Bulletin #77
• Screening tests should be offered to all women regardless of age
• Ideal screening test represents a combination of the best tests from the 1st and 2nd trimesters
FASTER Trial
• 15 U.S. centers • 38,189 singleton gestations • 117 with trisomy 21 • First and second trimester strategies compared
Malone et al., NEJM, 2005; 353:2001-11
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Integrated Screening • A combination of 1st and 2nd trimester screening tests • 11‐13 weeks: NT, PAPP‐A • 15‐17 weeks: AFP, hCG, estriol, & inhibin • Final result provided once Quad screen completed • All testing combined into a SINGLE result
FASTER Trial Detection Rate for 5% Screen Positive Rate 95
85 86 81
68 69 Integrated tests
NT Triple Quad NT + PAPPA + NT + PAPPA + Quad PAPPA + hCG Quad
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Screening Tests Detection rates based on 5% screen positive rate • First Trimester • First Plus Second – NT, PAPP‐A, hCG Trimester • 82‐87% – Integrated • Second Trimester • 95% – AFP, estriol, hCG, – Serum Integrated inhibin‐A • 85‐88% • 81%
FASTER , Malone et al, NEJM, 2005; 303:2001
Non‐Invasive Prenatal “Diagnosis”
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Cell‐free DNA in the Maternal Circulation Placenta Maternal plasma Maternal blood cells
•Cell-free fetal and cell-free maternal DNA circulate in maternal plasma as relatively short fragments (150-200 base pairs) •Fragments represent the entire genome •Placenta→ Fetal DNA •Blood cells→Maternal DNA •Fetal DNA: 5-25% (~10%) total cell-free DNA
Lo, Lancet 1997;350:485‐487
The Concept of Massively Parallel Shotgun Sequencing Relative amount of chromosome 21 Normal Mother Normal Fetus Normal Mother Down syndrome
18 copies + 2 copies 18 copies + 3 copies 20 copies 21 copies Need to distinguish 21 copies from 20 copies, a 5% difference. (assumes 10% of cfDNA is fetal)
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Schematic: Massively parallel genomic sequencing for fetal chromosomal aneuploidy
Chiu R W K et al. PNAS 2008;105:20458
%chr21 = 1 / 51 = 2%
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Data analysis
• 12 to 19 million sequences per sample matched (aligned) to the human genome • Unique matches on the first 25‐36 bp • Interpretation of results • Express percent of chromosome 21 matches to all matches • Express patient results in SD’s above/below expected
Percent unique reads and corresponding z‐score for chromosome 21 Higher proportion map to 21 with trisomy reads Blue normal male Orange normal female unique
Green T21 male all Red T21 female of
% score ‐ Z
Normal range
Chiu R W K et al. PNAS 2008;105:20458
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Laboratory Developed Tests • MPSS‐based tests • Sequenom Center for Molecular Medicine •Maternit21 PLUS™ •Introduced Fall, 2011 • Verinata Health •Verifi™ •March, 2012
Validation Studies:MaterniT21
• Multicenter study • 4,664 women • Blinded nested case‐control study • 212 Trisomy 21—105 <15 wk; 107 >15 wk • Seven euploid:each case—1,484 • z‐score at or above 3 diagnostic of T21 • Fetal fraction of cfDNA: 13.4% mean • Failure rate 0.8%
Palomaki et al, Genetics in Medicine, 2011; 13:913
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• T21 Detection • 209/212 98.6% (95% CI 95.9 – 99.7) • False‐Positive • 3/1,4710.2% (95% CI <0.1 –0.6)
• Failure and false‐negative associated with lower fetal fraction Palomaki et al, Genetics in Medicine, 2011; 13:913
Validation Studies: MaterniT21 PLUS • Same population • 62 Trisomy 18 • 12 Trisomy 13 • 3 euploid controls matched • z‐score at or above 3 diagnostic • Failure rate 0.9% • 3 failures T18
Palomaki et al, Genetics in Medicine, March 2012 14(3):296
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• Trisomy 18 • Detection 59/59 100% (95% CI 93.9 – 100) • Including failures 59/62 95.6% • False‐positive 5/1,688 0.3% (95% CI 0.1‐0.7) • Trisomy 13 • Detection 11/12 91.7% (95% CI 61 – 99) • False‐positive 16/1,688 0.9% (95% CI 0.5‐1.5)
Validation Studies:Verifi
• Whole chromosome aneuploidy • 2,882 women • Nested case‐control study (1:4 ratio) • 532 samples analyzed • Normalized chromosome values • > 4affected • <2.5 unaffected • 2.5 –4 indeterminate
Rava et al, SMFM abstract 837, AJOG Jan 2012 S367; Bianchi et al, Obstet Gynecol, 2012:119(5)
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Verifi Performance
Sensitivity 95% CI Specificity 95% CI Trisomy 21 100 (89/89) 95.9 – 100 100 (404/404) 99.1 – 100 (n=493) Trisomy 18 97.2 (35/36) 85.5 – 99.9 100 (460/460) 99.2 – 100 (n=496) Trisomy 13 78.6 (11/14) 49.2 – 99.9 100 (485/485) 99.2 – 100 (n=499) Female 99.6 97.6 – 99.9 99.5 (199/200) 97.2 – 99.9 (n=433) (232/233) Male 100 98.0 – 100 100 (249/249) 98.5 – 100 (n=433) (184/184) Monosomy X 93.8 (15/16) 69.8 – 99.8 99.8 (416/417) 98.7 – 99.9 (n=433)
Overall Performance: Verifi
• No false positives for autosomal aneuploidies • Mosaicism for trisomy 21 and monosomy X detected • Trisomy 20 and trisomy 16 detected • Sex chromosome aneuploidies classified
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LDT: Directed DNA Analysis
• Specific loci sequenced from chromosomes of interest • Less variability when applied • Increased accuracy, simple bioinformatics • Quoted shorter TAT and lower cost
Targeted DNA LDTs for NIPT
• Aria/Ariosa • Harmony™ • Introduced Summer 2012 • Natera • NATUS (next generation aneuploidy testing using SNPs) Panorama™ • Launched end 2012
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Harmony Non‐Invasive Chromosomal Evaluation (NICE) Study
• Locus‐specific oligonucleotides serve as sequencing templates • FORTE (Fetal‐fraction optimized Risk of Trisomy Evaluation) algorithm incorporates counts • LR based on fetal fraction, maternal age, GA • >1/100: High risk
Ashoor et al, AJOG 2012; 206:322; Norton et al, AJOG 2012; 207:137
NICE Results: T21 and T18
• T21 • Sensitivity 100% • False‐positive 0.03% • T18 • Sensitivity 97.4 % • False‐positive 0.07%
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Quantitative and Qualitative NIPT: Natera Panorama
• Fetal signal identified at polymorphic sites • Ratio fetal:maternal SNPs at target and reference chromosomes • Individualized trisomy risk assessment for each chromosome based on statistical genetics
Zimmermann et al, Prenatal Diagnosis 2012; 32:1233:
Result Comparisons
Detection Rate Sequenom Verinata Ariosa Natera False Positive Rate
98.6‐99.1% <99.9% 100% >99% Trisomy 21 0.2% 0.2% 0.1% 0.0%
100% 97.4% 98% >99% Trisomy 18 0.3% 0.4% 0.1% 0.0%
91.7% 87.5% 80% >99% Trisomy 13 1.0% 0.1% 0.05% 0.0% 45,X Not 95% >99% Not evaluated (Monosomy X) evaluated 1.0% 0.0%
Based on available published data as of Feb 2013
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Fetal Fraction Key in Success
• Affected by: • Gestational age—9‐10 weeks • Obesity • Other??? • Average 10‐15% • Equivocal or no call rate if <5%
Application of NIPT
• Role in clinical practice • Screening vs. diagnosis • Applicable population • Capacity/Infrastructure/TAT • Insurance coverage
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Reflex Test Modeling
• Using as MPSS secondary screening test vs invasive testing • Reduce diagnostic testing by >90% • Reduce fetal loss rate by 93‐95% • Improve trisomy detection 3‐6%
Palomaki et al, Genetics in Medicine, March 2012 14(3):296
• NOT for low-risk, general population women • Appropriate Populations: • AMA • Prior affected • Abnormal screen • Ultrasound abnormality • Robertsonian translocation involving 13 or 21 • Pre- and post-test counseling on false positives and false negatives • Need more information for multiples
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Diagnostic Testing
Deletions& Duplications: Submicroscopic
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FISH: Targeted for Microdeletion Syndromes
• Caused by deletion of submicroscopic amounts of DNA • Involves multiple genes • DiGeorge Syndrome 22q11 • Williams Syndrome 7q11
Array Comparative Genomic Hybridization (CGH)
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A Matter of Resolution: How close are we focused on changes?
• G‐band chromosomes • 5‐15 Mb level • FISH • 2 Mb level
Advantages of Chromosomal Microarray • Finer resolution • 1 Mb, 100 Kb or lower • Multiple areas simultaneously • DNA based • Living cells not required
Albertson et al, Hum Mol Gen, 2003; Oostlander et al, Clin Gen, 2004
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Limitations of Chromosomal Microarray
• Cannot identify balanced translocations • Potential to detect variants of unknown significance (VOUS)
Microarray: Postnatal Genetic Diagnosis • Developmental delay, autism spectrum, or multiple anomalies • 3% abnormal G‐banded chromosomes • 15‐20% abnormal array • ACMG: Array CGH first line for postnatal evaluation of DD, ASD, MCA
Devries et al, 2005; Friedman et al, 2006; Menten et al, 2006; Miller et al, AJHG, 2010
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Study Procedures • Indications: • AMA • Abnl screen • US anomaly • Prior history/anxiety • Risks • Possibility of findings of uncertain significance • ID variants associated with adult‐onset disease
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Study Procedures • Outcomes • Success rate of microarray • Ability to ID common autosome and sex‐chromosome aneuploidies • Prevalence of CNVs with normal karyotype • Ability to ID uncommon cytogenetic abnormalities (markers, rearrangements, etc)
Study Procedures • Arrays covered: • Regions of known disease loci • Pericentromeric regions • Subtelomeres • Genomic backbone 75kb
• SNP based array used at some labs as well
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Study Procedures • Positive Results • CNV in or overlapping a targeted region • ≥1 Mb CNV in other region • <1 Mb but includes a gene implicated in a chromosome disorder, AD condition, or X‐linked disorder
Study Procedures
• Pathogenic—well‐described phenotype • Benign—based on databases and publication • Uncertain Significance • Likely benign—small size, gene content, normal US, negative family hx • Uncertain—subjected to adjudication
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Results • 4,406 women enrolled • Microarray result: 98.8%
• Detected all autosomal and sex chromosome aneuploidies • Did not detect: • Balanced rearrangements • Triploidy • 1 of 3 marker chromosomes
Results • 1399 CNVs identified • 1234 (88%) common benign • 35 (2.5%) known pathogenic • 130 (9.8%) uncertain significance • After adjudication, 61 felt to be significant enough to report
• 96 potential pathogenic (6.8%)
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Frequency of Additional Information
Indication N Pathogenic 95% CI Any 3822 2.5% 2.1 – 3.1 AMA 1966 1.7% 1.2 – 2.4 Abnormal Screen 729 1.6% 0.9 – 2.9
Anomaly on US 755 6.0% 4.5 – 7.9
Other 372 1.3% 0.6 – 3.1
Microarray and Stillbirth • 532 stillbirths • Result: Microarray 87% vs karyotype 70% • Aneuploidy and CNV—8.3% vs 5.8% • With anomalies 30% vs 19%
• Microarray increased information in: • 42% all stillbirths • 35% antepartum stillbirths • 54% stillbirths with anomalies
Reddy et al, NEJM, December 2012 367:2185
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Role of Microarray
• Benefit of additional information • Risk of additional information • Insurance coverage • Appropriate patients • Anomalies • Others?
Summary
• NIPT is able to provide detection of common aneuploidies with high sensitivity and specificity • CGH array can provide additional information beyond that of normal karyotype, especially in the setting of anomalies or stillbirth • Pre‐ and post‐test counseling regarding the limitations, benefits, and risks of each type of testing is vital
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Questions?
Improvements with recent advances • 3.4% with normal karyotype‐‐CNVs with uncertain clinical significance • Based on 2012 data, only 1.5% would remain of uncertain significance • SNP arrays will detect triploidy
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Landmark papers
PNAS 2008;105:15255
PNAS 2008;105:20458
2.01 2.00 (2.11 – 2.01) 1.98 2.11 2.02 Z = ------= 5.0 2.03 0.02 1.99 ------Higher proportion of sequenced fragments map 2.01 (sd=0.02) to Ch21 in setting of trisomy—Higher z-score
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Arrays in Prenatal Settings
• Schaeffer et al, AJHG, 2004 • sAb n=41 • 4 additional abnormalities • Benkhalifa et al, Prenatal Dx, 2005 • sAb with failed culture n=26 • 15/26 with chromosome imbalances • LeCaignec et al, J Med Gen, 2005 • Fetuses with multiple malformation and normal karyotype n=49 • 10% with imbalance
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