Diagnóstico prenatal no invasivo en sangre materna

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Ponencia: Diagnóstico prenatal no invasivo en sangre materna. …

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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  • 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  
  • 7. Aumento de DNA Fetal del Cr.21 en Plasma Materno
  • 8. Fetal Trisomy Detection with cfDNA Extra fragments derived from fetal trisomy 21 Fetal cfDNA Maternal cfDNA Reference chromosome Chromosome 21 fragments
  • 9. 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  
  • 10. Fetal Fraction Constant Across Risk Groups Brar H et al., J Matern Fetal Neonatal Med. 2013 Jan;26(2):143-5.
  • 11. 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.
  • 12. 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.
  • 13. 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
  • 14. 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
  • 15. 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
  • 16. 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  
  • 17. 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
  • 18. 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  
  • 19. 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
  • 20. 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
  • 21. 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  
  • 22. 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
  • 23. Average Risk Study       Am J Obstet Gynecol. 2012 Nov;207(5):374.e1-6 (Epub 2012 Sep 19)
  • 24. 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
  • 25. 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
  • 26. 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
  • 27. Risk  comparison: Combined Screening vs NIPT 1st Trimester Combined Screening
  • 28. 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  
  • 29. 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
  • 30. Screening for T21, 18 and 13 by cfDNA in Maternal Plasma SCRENNING 22% HISTORY 3% ANXIETY 22% FIV 8% US 13% MAT.AGE 32%
  • 31. 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%)
  • 32. 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
  • 33. vincenzo.cirigliano@labco.eu