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Ponencia: Diagnóstico prenatal no invasivo en sangre materna. Dr. Vicenzo Cirigliano. Responsable de genética molecular y coordinador de diagnóstico prenatal Labco Diagnosis. Barcelona
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Vincenzo Cirigliano PhD
Genética Molecular y Unidad de Diagnóstico Prenatal
Labco, Barcelona vincenzo.cirigliano@labco.eu
Cribado de Anomalías Cromosómicas:
DNA Fetal en Plasma Materno
- Análisis Citogenético necesita ≈ 2 Semanas
- Aneuploidías de X, Y, 13, 18 y 21
≈ 95% de Anomalías Cromosómicas
- Las Indicaciones Han Cambiado
- Cribado en el 1er trimestre
Diagnostico en el Segundo?!
Diagnóstico Prenatal de Anomalías Cromosómicas
COUNSELLING (MATERNAL AGE/PREVIOUS HISTORY)
CVS / AMNIOCENTESIS
QF-PCR CYTOGENETICS
SEQUENCES OF PRENATAL TESTS
Non Invasive Screening 1st / 2nd SERUM - ULTRASOUND
aCGH
- Falsos Positivos Técnicas invasivas innecesarias, angustia
- Tiempo Puede extenderse al segundo trimestre - Conveniencia Múltiples visitas y ecografía limitan acceso/eficacia - Seguridad
Rechazo a técnicas invasivas por el riesgo de perdida fetal
Limitaciones del Cribado Actual
§ 1997: Secuencias del cromosoma Y detectadas en plasma y suero de gestantes con fetos masculinos (Lo et al 1997)
§ 1998: Cuantificación del cfDNA por Real-Time PCR § Porcentaje de cfDNA superior en Plasma
§ Early pregnancy: 0.4 – 11.9% (mean 3.4%)
§ Late pregnancy: 2.3 – 11.4% (mean 6.2%) § RNA placenta específico (ZFY) en plasma materno (Poon et
al. 2000)
§ ffDNA desde el trofoblasto (Alberry et al. 2007)
DNA Fetal en Plasma Materno
Cell-free DNA en Sangre Materna • En todas las gestaciones hay cfDNA de madre y feto en circulación materna • Cell-free DNA (cfDNA) es presente en fragmentos muy cortos • La cantidad de cfDNA fetal solo es una pequeña fracción del cfDNA materno
Aumento de DNA Fetal del Cr.21 en Plasma Materno
Fetal Trisomy Detection with cfDNA
Chromosome 21 fragments
Reference chromosome
Fetal cfDNA
Maternal cfDNA
Extra fragments derived
from fetal trisomy 21
Fetal Trisomy Detection with cfDNA
Chromosome 21 fragments
Reference chromosome
Fetal cfDNA
Maternal cfDNA
Fetal Frac)on
Expected ra)o for Trisomy
4% 1.02
10% 1.05
20% 1.10
40% 1.20
Including fetal fraction in analysis improves accuracy of result
Fetal Fraction Constant Across Risk Groups
Brar H et al., J Matern Fetal Neonatal Med. 2013 Jan;26(2):143-5.
Massively Parallel Shotgun Sequencing (MPSS)
• MPSS is a random sampling of cfDNA fragments • An arbitrary z-score value is used as a cut-off for trisomy
Palomaki et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med. 2011 Nov;13(11):913-20.
N=1696
MPSS Unclassified Values
• “Unclassified” zone for values between 2.5-‐4
• DisproporAonate number of posiAves in this zone
Bianchi, DW, et al. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol. 2012 May;119(5):890-901.
MPSS Performance
40% 60% 80% 100%
T21 (99-100%)
T18 (84-100%)
T13 (44-100%)
0-2%
0-2%
0-6%
Detection Rate False Positive Rate
Palomaki GE, Kloza EM, Lambert-Messerlian GM, et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med 2011 Nov;13(11):913–20.; Bianchi DW, Platt LD, Goldberg JD, Abuhamad AZ, Sehnert AJ, Rava RP, Genome-Wide Fetal Aneuploidy Detection by Maternal Plasma DNA Sequencing. Obstet Gynecol. [Epub ahead of print] 2012 Feb 22.; Chiu et. Al BMJ 2011;342:c7401 Chen et.al (2011) http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021791
• El cariotipo de la placenta no siempre refleja el fetal • Más frecuente para Chr 13 y 18 que Chr 21
cfDNA se origina en placenta " Citotrofoblasto " Paragonable a un “cariotipo semidirecto” " CPM podrían generar falsos negativos y falsos
positivos, en particular para T13 y T18
T18, T13 y Mosaicismos Confinados a Placenta
TARGETED NIPT 21, 18, 13
(Digital ANalysis of Selected Regions)
• Directed assay for cfDNA isolation and analysis. • Targeted method allows for high throughput DNA sequencing
High throughput and scalable test Clinically interpretable results to patients
DANSRTM FORTETM
(Fetal-fraction Optimized Risk of Trisomy Evaluation)
" New analysis that provides a trisomy risk score " Incorporates DANSR assay results (chromosome counts, fetal fraction), maternal and gestational age
Chr 21, 18, 13 cfDNA Other Chr cfDNA
Unmapped cfDNA
cfDNA in blood
MPSS (shotgun)
More efficient
Random analysis of cfDNA
Assay Comparison – Targeted vs MPSS
DANSR™ (Directed)
Low False Positives
False positive rate List price
T21 T18 T13 Y Total
<0.1% <0.1% <0.1% N/A <0.3% $795
0.2% 0.28% 0.97% 0.6% 2.0% ~$2,700
1. Norton et al, Am J of Obstet and Gyn, 2012; 2. Nicolaides KH et al, Am J Obstet Gynecol 2012; 3. Ashoor G et al., Ultrasound Obstet Gynecol 2012 (online); 4. Palomaki GE et al, Genet Med 2011; 5. Palomaki et al, Genet Med 2012; 6. MaterniT21 report example accessed Aug 2012
1-3
Targeted NIPT shows false positive rates 5-7x lower than MPS
Maternal weight effects -‐ commercial data 22,000 Wang et al., Prenat Diag (in press)
Consistent in high and low-‐risk women 3,007 Brar et al, J Mat Fet Neonat Med 2013
Maternal weight and fetal factors, study 2 1,949 Ashoor et al. Ultras Obstet Gyn 2013
Maternal weight and fetal factors, study 1 400 Ashoor et al., Fetal Diagn Ther 2012
Study Subjects Reference
NICE -‐ Cohort validaAon study 3,228 Norton M et al., AJOG 2012
General screening populaAon, 1st trimester 2,049 Nicolaides et al, AJOG 2012
Trisomy 13 1,949 Ashoor et al., Ultra Obstet Gyn 2013
Kypros Nicolaides clinical implementaAon 701 Mar Gil et al, Ultra Obstet Gyn (in press)
EU-‐NITE -‐ European study 520 Verweij et al., (submi`ed)
High-‐risk populaAon, 1st trimester 400 Ashoor et al., AJOG 2012
FORTE 338 Sparks et al., AJOG 2012
DANSR 298 Sparks et al., Prenat Diagn 2012
Ob/Gyn real world experience 289 Fairbrother et al., Prenat Diagn 2013
Clinical Validity and Use
Fetal Frac)on
Validacion/Aplicación Clínica
NICE Study " 50 participating clinical sites in U.S. and Europe " Largest cohort study to date – All eligible subjects evaluated " Study population was women undergoing invasive testing for any indication
and thus included low risk women
Sensitivity Specificity False Positive Rate
Trisomy 21 100% (81/81)
99.97% (2887/2888)
0.03% (1/2888)
Trisomy 18 97% (37/38)
99.93% (2886/2888)
0.07% (2/2888)
Norton ME et al. (2012) American Journal of Obstetrics and Gynecology
Harmony vs other NIPT tests
Harmony MaterniT21+ (Sequenom) verifi (Verinata) NIFTY
(BGI) PraenaTest (Lifecodexx)
Panorama (Natera)
Medición % DNA Fetal + + _ _ + +
% de Éxito + + + + + _
Ovodonación / Gemelares + _ + _ _ _
Estudios Clínicos Publicados + + _ _ _ _
Precio 695 _ 1.000 820 _ 900
Overall cfDNA Screening Performance
Detection rate FPR
Trisomy 21 590 / 594 (99.5%) 0.1%
Trisomy 18 222 / 230 (97%) 0.1%
Trisomy 13 30 / 38 (79%) 0.1%
Chiu et al, 2011; Chen et al, 2011; Ehrich et al, 2011; Palomaki et al, 2011; Bianchi et al, 2012; Sparks et al, 2012; Ashoor et al, 2012; Norton et al,
2012
cfDNA analysis does not always correlate with fetal genotype
Trisomy 21 N=200 99.800 Normal
METHOD OF SCREENING DR Detected False Positive
Serum biochemistry at 16 wks 70% 140 5% 4990
Combined test at 12 wks 90% 180 5% 4990
Combined plus at 12 wks 97% 194 3% 2994
Cell-free DNA >99% >199 <0.1% <100
100.000 Pregnancies
Screening for Aneuploidies by cfDNA in maternal blood
By Far the best available option for T21 and 18
· Can be offered to all women irrespective of risk · Can provide result in the 1st trimester of pregnancy K. Nicolaides SMFM SF 2013
Am J Obstet Gynecol. 2012 Nov;207(5):374.e1-6 (Epub 2012 Sep 19)
Average Risk Study
10 11 16 15 14 13 12 20 19 18 17
CVS AMNIO
ANATOMY U/S LOW RISK
HIGH RISK
U/S withNT
NIPT
• high risk NIPT • increased NT • abnl U/S
• karyotype • microarray
Screen alternative: NIPT + 1st trimester ultrasound
10 11 16 15 14 13 12 20 19 18 17
CVS AMNIO
Screen alternative: NIPT + 1st trimester ultrasound
ANATOMY U/S LOW RISK
HIGH RISK
U/S withNT
NIPT
• high risk NIPT • increased NT • abnl U/S
• karyotype • +/- microarray
Up to 10% of NT > 3.5mm with normal karyotype has CNV on array
Implementation of maternal blood cfDNA testing in early screening for aneuploidies
• high risk cfDNA • Fetal defects • NT >3.5 mm
CVS
10 weeks: • Scan to measure the
fetus • Blood for Harmony test-‐
shipped to California • Blood for combined test
12 weeks: • Detailed ultrasound scan • Nuchal Translucency • Discuss results • Decide if CVS is necessary
Gil, Quezada, Bregant, Ferraro, and Nicolaides. Ultr Obstet Gyn, 2013
Risk comparison: Combined Screening vs NIPT
1st Trimester Combined Screening
Alternative to 1st trimester screening : Conclusions
• NIPT with cfDNA – substanAal reducAon in false posiAve test results as compared to 1st trimester combined screening in a general pregnancy populaAon
– Greater separaAon of high and low risk esAmates over a range of risk cut-‐offs
– Expect easier decision making regarding invasive dx
• NIPT for aneuploidy screening in the general pregnant populaAon could help to reduce unnecessary invasive procedures and maternal anxiety
Ø 24h National Logistics - 48h International Shipping
Ø Cell-Free DNA BCT STRECK tubes (2x10mL)
Ø 5 Days limit from sampling to accession
Ø 8 Working Days Report – Sampling to Report 2 weeks
Introduction of cfDNA Screening in Spain
US 13%
MAT.AGE 32% ANXIETY
22%
SCRENNING 22%
HISTORY 3%
FIV 8%
Screening for T21, 18 and 13 by cfDNA in Maternal Plasma
4867 Samples
14 Days 98%
21 Days 2%
No result 179 (3.7%)
Result 75 (62%)
Tested 4819 (99%)
Not suitable (n=48)
Result 4640 (96.3%)
Redraw 121
No result 46 (38%)
Result 5 No result 7
2ndRedraw 12
Harmony Test: Clinical Application
Normal n= 4283 (92%)
Trisomy 21 n= 54 (1,1%)
Trisomy 18 n= 5
Trisomy 13 n= 3
XY Chr. n= 32 (0,6%)
Ø Low false positives
Ø Powerful Complement or Alternative to Conventional Screening
Ø Reduce unnecessary testing in selected indications
Ø Awaited from patients
Ø Widespread limited by cost
Introduction of cfDNA Screening in Spain
vincenzo.cirigliano@labco.eu
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