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