Diagnóstico Prenatal de Anomalías Cromosómicas Nuevos Métodos, Nuevas Indicaciones

Transcripción

1 Diagnóstico Prenatal de Anomalías Cromosómicas Nuevos Métodos, Nuevas Indicaciones 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 QF-PCR en Diagnóstico Prenatal 99.9% Detección: -Edad Materna -Riesgo Bioquímico -Angustia 85% de Casos 95-97% Detección: TN Aumentada Marcadores Ecográficos 15% de Casos V.Cirigliano et.al. (2005) Reprod Biomed Online. 11(6):671-3

4 QF-PCR as Stand Alone Screening (1 st Combined - Ultrasound) Abnormal Ultrasound Family History Karyotype acgh NT<3mm or Risk >1:250, Maternal Age Patient s Choice or Targeted Sweden, Finland Norway, Holland UK

5 Cytogenetic s Chromosome Analysis trisomy 21 deletion 11q Prader-Willi syndrome Molecular Cytogenetics FISH CGH Array-CGH deletion 8p deletion 12q 322 genes >40 genes >30 genes >50 genes >15 genes 46 Mb ca. 10 Mb 4 Mb ~ 8 Mb ~ 1,5 Mb

6 CytoChip Focus BAC Array o 3380 Clones Triplicate/quadruplicate o Genome Backbone 1Mb Resolution o 139 Disease Loci 100kb Resolution o BAC Clones Selection/Validation - Avoid Known CNVs - Flow-sorted chromosomes hybs - FISH mapped - Run on > Clinical Samples

7 Nuevas Perspectivas en DP Marcadores ecograficos, QF-PCR y cariotipo normal/array-cgh Con los Arrays se pueden detectar anomalias que no son objecto de cribaje Array-CGH a todas las indicaciones de tecnicas invasivas? Transición hacia nuevas indicaciones Complejidad del Asesoramiento Genético

8 SEQUENCES OF PRENATAL TESTS COUNSELLING (MATERNAL AGE/PREVIOUS HISTORY) Non Invasive Screening 1 st / 2 nd SERUM - ULTRASOUND CVS / AMNIOCENTESIS QF-PCR acgh CYTOGENETICS

9 - Falsos Positivos Técnicas invasivas innecesarias, angustia - Tiempo Puede extenderse al segundo trimestre - Conveniencia Múltiples visitas y ecografía limitan acceso/ eficacia - Seguridad Limitaciones del Cribado Actual Rechazo a técnicas invasivas por el riesgo de perdida fetal

10 Cell-free DNA en Sangre Materna Cell-free DNA (cfdna) es presente en fragmentos muy cortos En todas las gestaciones hay cfdna de madre y feto en circulación materna La cantidad de cfdna fetal solo es una pequeña fracción del cfdna materno cfdna desaparece en pocas horas despues del parto

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12 Fetal Trisomy Detection with cfdna Fetal cfdna Maternal cfdna Reference chromosome Chromosome 21 fragments

13 Fetal Trisomy Detection with cfdna Fetal cfdna Extra fragments derived from fetal trisomy 21 Maternal cfdna Reference chromosome Chromosome 21 fragments

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 Nov; 13(11):

15 MPSS Unclassified Values Unclassified zone for values between Disproportionate number of positives in this zone Bianchi, DW, et al. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol May;119(5):

16 MPSS Performance Detection Rate False Positive Rate T21 (99-100%) 0-2% T18 (84-100%) 0-2% T13 (44-100%) 0-6% 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): ; 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)

17 TARGETED NIPT 21, 18, 13 DANSR TM (Digital ANalysis of Selected Regions) Directed assay for cfdna isolation and analysis. Targeted method allows for high throughput DNA sequencing FORTE TM (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 High throughput and scalable test Clinically interpretable results to patients

18 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

19 Validacion/Aplicación Clínica Study Status Description NICE (Non-Invasive Chromosomal Evaluation) Average Risk (Nicolaides) Ariosa Blinded Nicolaides Blinded Proof of Concept Trisomy 13 Fetal Fraction NICE substudy Fetal Fraction NITE (Non-Invasive Trisomy Evaluation) NEXT (Non-invasive EXamination of Trisomy) Published Editor s choice in The Gray Journal (August 2012) Published The Gray Journal (2012, avail online) Published Editor s choice in The Gray Journal (April 2012) Published Editor s choice in The Gray Journal (April 2012) Published cover article Prenatal Diagnosis (Jan 2012) Published The White Journal (2012, avail online) Published J Mat Fet Med (2012, avail online) Published Fetal Diagnosis and Therapy (2012) Enrolled Enrolling 19 Multi-center (50 sites) clinical validation study, combined high risk and low risk women. Largest NIPT cohort study. Exclusive average-risk study of Harmony test in 1 st trimester pregnancy Blinded study with risk score reporting 1 st trimester blinded study Initial description of directed cfdna approach with combined average-risk and high-risk women Performance for T13 detection with combined averagerisk and high-risk women Fetal fraction same in high-risk and low-risk women Fetal fraction correlated to placental mass Multi-center European blinded study Multi-center blinded study of average risk women comparing Harmony to 1 st trimester combined screening

20 NICE Study

21 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 % (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

22 Average Risk Study

23 Independent blinded study Patient population: Average Risk Study 1st trimester pregnancy (11-13 weeks gestation) General screening population of 2,049 women Results NIPT test detected all trisomy cases Trisomy 21: 8 of 8; Trisomy 18: 2 of 2 Risk score of >99% given for each trisomy False positive rate NIPT: 2 of 1,939 (0.1%) No false positives for trisomy % false positives for trisomy 18 Conventional screening (serum + NT ultrasound): 87 of 1,939 (4.5%) Nicolaides KH et al. (2012) American Journal of Obstetrics and Gynecology

24 Average Risk Study Risk Score Comparison Both figures have the same number of patients 10 Trisomies 1,939 Normal 1 st trimester screening NIPT Trisomy False positive Non-trisomy FPR: 4.5% FPR: 0.1% Trisomy Risk (%) ,1 Trisomy risk (%) ,1 0, , Fetal Fraction (%) Fetal Fraction (%)

25 Potencial Utilidad Clínica del NIPT Cribado Precoz Después del Cribado En Sustitución de AF-CVS NIPT + Ecografía: Probable mejora de la eficiencia del cribado actual NIPT detecta las trisomías comunes con precisión Ecografía centrada en anomalías no relacionadas a las trisomías Descartar trisomías mas frecuentes con elevada especificidad NIPT solo detecta 3 de las posibles anomalías cromosómicas Técnica invasiva necesaria de todas formas para confirmar eventuales No es un test resultados diagnostico, positivos resultados de riesgos van confirmados con técnicas invasivas Solo útil para descartar trisomías mas frecuentes La utilización de los arrays ha ampliado enormemente el poder diagnóstico de las técnicas invasivas

26 Screening for Aneuploidies by cf-dna in maternal blood By Far the best available option for T21 and Pregnancies Trisomy 21 N= 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 Can be offered to all women irrespective of risk Can provide result in the 1 st trimester of pregnancy

27 Low False Positives False positive rate T21 T18 T13 Y Total List price 1-3 <0.1% <0.1% <0.1% N/A <0.3% $ % 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

28 SEQUENCES OF PRENATAL TESTS COUNSELLING (MATERNAL AGE/ HISTORY) Non Invasive Screening 1 st Trimester mid/low NIPD NIPD? High/Ultrasound CVS / AF Confirmations CVS / AF QF-PCR QF-PCR acgh? CYTOGENETICS

29 SEQUENCES OF PRENATAL TESTS COUNSELLING (MATERNAL AGE/ HISTORY) Low Risk Screening (Anxiety) NIPD Common Trisomies Residual risk 1:1200 CVS / AF QF-PCR acgh Whole Genome

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31 QF-PCR Como Único Test Diagnóstico? Cribado 1º Trimestre/Ecografía Técnicas invasivas en gestaciones de riesgo QF-PCR en Todos los Casos Análisis Citogenético sólo en: Marcadores Ecográficos (Array-CGH) Historia Familiar V.Cirigliano et.al. (2009) Prenat Diagn. 29(1):40-9