Diagnóstico prenatal no invasivo en sangre materna
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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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Diagnóstico prenatal no invasivo en sangre materna
- 1. Cribado de Anomalías Cromosómicas: DNA Fetal en Plasma Materno Vincenzo Cirigliano PhD Genética Molecular y Unidad de Diagnóstico Prenatal Labco, Barcelona vincenzo.cirigliano@labco.eu
- 2. Diagnóstico Prenatal de Anomalías Cromosómicas - 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?!
- 3. SEQUENCES OF PRENATAL TESTS COUNSELLING (MATERNAL AGE/PREVIOUS HISTORY) Non Invasive Screening 1st / 2nd SERUM - ULTRASOUND CVS / AMNIOCENTESIS QF-PCR aCGH CYTOGENETICS
- 4. Limitaciones del Cribado Actual - 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
- 5. DNA Fetal en Plasma Materno § 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)
- 6. 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
- 9. Aumento de DNA Fetal del Cr.21 en Plasma Materno
- 11. Fetal Trisomy Detection with cfDNA Extra fragments derived from fetal trisomy 21 Fetal cfDNA Maternal cfDNA Reference chromosome Chromosome 21 fragments
- 12. Fetal Trisomy Detection with cfDNA Including fetal fraction in analysis improves accuracy of result Fetal cfDNA Fetal Frac)on 4% 1.10 40% Chromosome 21 fragments 1.05 20% Reference chromosome 1.02 10% Maternal cfDNA Expected ra)o for Trisomy 1.20
- 13. Fetal Fraction Constant Across Risk Groups Brar H et al., J Matern Fetal Neonatal Med. 2013 Jan;26(2):143-5.
- 14. 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 N=1696 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.
- 15. 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.
- 16. MPSS Performance False Positive Rate Detection Rate T21 0-2% (99-100%) T18 0-2% (84-100%) T13 0-6% (44-100%) 40% 60% 80% 100% 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
- 17. T18, T13 y Mosaicismos Confinados a Placenta • 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
- 18. TARGETED NIPT 21, 18, 13 DANSR FORTE TM TM (Digital ANalysis of Selected Regions) (Fetal-fraction Optimized Risk of Trisomy Evaluation) • Directed assay for cfDNA isolation and analysis. • Targeted method allows for high throughput DNA sequencing " New analysis that provides a trisomy risk score " Incorporates DANSR assay results (chromosome counts, fetal fraction), maternal and gestational age High throughput and scalable test Clinically interpretable results to patients
- 19. Assay Comparison – Targeted vs MPSS cfDNA in blood Chr 21, 18, 13 cfDNA Other Chr cfDNA Unmapped cfDNA DANSR™ (Directed) More efficient MPSS (shotgun) Random analysis of cfDNA
- 20. Low False Positives False positive rate List price T21 T13 Y Total <0.1% <0.1% <0.1% N/A <0.3% $795 0.2% 1-3 T18 0.28% 0.97% 0.6% 2.0% ~$2,700 Targeted NIPT shows false positive rates 5-7x lower than MPS 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
- 21. Validacion/Aplicación Clínica 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 Maternal weight effects -‐ commercial data Fetal Frac)on Reference NICE -‐ Cohort validaAon study Clinical Validity and Use Subjects 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
- 22. 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 Trisomy 21 Trisomy 18 Specificity False Positive Rate 100% 99.97% 0.03% (81/81) (2887/2888) (1/2888) 97% 99.93% 0.07% (37/38) (2886/2888) (2/2888) Norton ME et al. (2012) American Journal of Obstetrics and Gynecology
- 23. Harmony vs other NIPT tests Harmony Medición % DNA Fetal + MaterniT21+ (Sequenom) verifi (Verinata) NIFTY PraenaTest (Lifecodexx) Panorama + _ _ + + (BGI) (Natera) % de Éxito + + + + + _ Ovodonación / Gemelares + _ + _ _ _ _ _ _ 820 _ 900 Estudios Clínicos Publicados + + _ Precio 695 _ 1.000
- 24. 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% cfDNA analysis does not always correlate with fetal genotype 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
- 25. Screening for Aneuploidies by cfDNA in maternal blood By Far the best available option for T21 and 18 METHOD OF SCREENING Serum biochemistry at 16 wks Combined test at 12 wks Combined plus at 12 wks Cell-free DNA 100.000 Pregnancies Trisomy 21 N=200 DR Detected 70% 90% 97% >99% 99.800 Normal False Positive 140 5% 4990 180 5% 3% <0.1% 4990 2994 <100 194 >199 · Can be offered to all women irrespective of risk · Can provide result in the 1st trimester of pregnancy K. Nicolaides SMFM SF 2013
- 26. Average Risk Study Am J Obstet Gynecol. 2012 Nov;207(5):374.e1-6 (Epub 2012 Sep 19)
- 27. Screen alternative: NIPT + 1st trimester ultrasound U/S with NT NIPT HIGH RISK 10 11 12 • • • 13 ANATOMY U/S LOW RISK high risk NIPT increased NT abnl U/S 14 15 16 CVS 17 18 AMNIO • karyotype • microarray 19 20
- 28. Screen alternative: NIPT + 1st trimester ultrasound U/S with NT NIPT HIGH RISK 10 11 12 • • • 13 LOW RISK high risk NIPT increased NT abnl U/S 14 15 16 Up to 10% of NT > 3.5mm with normal karyotype has CNV on array 17 CVS 18 AMNIO • karyotype • +/- microarray 19 ANATOMY U/S 20
- 29. Implementation of maternal blood cfDNA testing in early screening for aneuploidies 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 • high risk cfDNA • Fetal defects • NT >3.5 mm Gil, Quezada, Bregant, Ferraro, and Nicolaides. Ultr Obstet Gyn, 2013
- 30. Risk comparison: Combined Screening vs NIPT 1st Trimester Combined Screening
- 31. 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
- 32. Introduction of cfDNA Screening in Spain 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
- 33. Screening for T21, 18 and 13 by cfDNA in Maternal Plasma SCRENNING 22% HISTORY 3% ANXIETY 22% FIV 8% US 13% MAT.AGE 32%
- 34. Harmony Test: Clinical Application 4867 Samples Not suitable (n=48) Tested 4819 (99%) Result 4640 (96.3%) No result 14 Days 98% 179 (3.7%) Redraw 121 21 Days 2% No result 46 (38%) Normal n= 4283 (92%) Trisomy 21 n= 54 (1,1%) Trisomy 18 n= 5 Trisomy 13 n= 3 XY Chr. n= 32 (0,6%) 2ndRedraw 12 No result 7 Result 5 Result 75 (62%)
- 35. Introduction of cfDNA Screening in Spain 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