Clinical implementation of maternal blood cell-free DNA testing in first trimester screening for aneuploidies

CLINICAL IMPLEMENTATION OF MATERNAL BLOOD CELL-FREE DNA TESTING IN FIRST TRIMESTER SCREENING FOR ANEUPLOIDIES

Submitted by

María del Mar Gil Mira

For the degree of International Doctor in Medicine

Universidad Autónoma de Madrid September 2015

Directors:

Professor Kypros Herodotou Nicolaides King’s College University of London, England Professor José Luis Bartha Rasero

Universidad Autónoma de Madrid, Spain

Professor Kypros Herodotou Nicolaides King’s College Hospital School of Medicine London, England

I confirm that Dr María del Mar Gil Mira has carried out under my supervision the studies presented in the Thesis: Clinical implementation of maternal blood cell-free DNA testing in first trimester screening for aneuploidies.

I have read the Thesis and I am happy for this to be presented to the Tribunal for The Degree of International Doctor in Medicine.

Professor Kypros Herodotou Nicolaides

London September 2015

Professor José Luis Bartha Rasero Universidad Autónoma de Madrid, Spain

I confirm that Dr María del Mar Gil Mira has carried out under my supervision the studies presented in the Thesis: Clinical implementation of maternal blood cell-free DNA testing in first trimester screening for aneuploidies.

I have read the Thesis and I am happy for this to be presented to the Tribunal for The Degree of International Doctor in Medicine.

Professor José Luis Bartha Rasero

Madrid September 2015

ACKNOWLEDGEMENTS

My deepest gratitude is to Professor Nicolaides, who has shared with me his dream of “changing the world”, his knowledge, passion and enthusiasm for fetal medicine, scientific research and the work properly done, since the first time we met. These three years working by your side have been a whole lesson of life and you have been my counsellor in both, professional and personal decisions.

Thank you, not only for directing my thesis, but also for giving me the opportunity to believe in my dream and, more importantly, the courage and the tools to fight to achieve it.

I am also very grateful to Professor Bartha, who guided me at the beginning of my way to be trained in fetal medicine and also during my first steps towards research. Thank you for your support in the distance all these three years. I am very pleased that you are co-directing this thesis with Professor Nicolaides.

Thank you very much to Rocío for so many unforgettable moments during our long working hours. You have been not only the best mate but you also became the best friend. I am also very glad that Alex, Karen and Konstantinos were also chosen to be running the study with me, because they have been perfect in their work. I believe that I have been tremendously fortunate to have worked with these four outstanding individuals and scholars, and I am deeply appreciative to each of them for their important contributions.

I also want to thank all the co-authors of the studies, especially Liona, Ranjit and Sol, whose knowledge, energy and enthusiasm were critical to this effort.

And of course, all the fellows scanning, collecting data, recruiting patients and getting outcomes. Without your hard work the completion of these studies would not have been possible. I learnt a lot from all of you.

I am very grateful to The Fetal Medicine Foundation, which founded all the studies included in this thesis and helps every year hundreds of doctors like me, make their dreams become true. To Ariosa Diagnostics Inc., for analysing the samples from our studies. And to Fundación Alfonso Martín Escudero, which believes that young Spanish researchers have a lot to do, and helps us not have to worry about anything else than working well.

A special word of thanks also goes to my beloved family, who has been a constant source of love, concern, support and strength all these years. I know I always have my family to count on when times are rough. I hope I have made you proud.

And finally, I am thankful to Jose, who has made all the difference in my life and

to whom this thesis is dedicated. Words cannot express my gratitude for

everything you have done. Specially, for believing in my project and supporting

me, even if this meant staying far away from each other. Thank you for

accompanying me on this adventure, I look forward to our next one!

AGRADECIMIENTOS

Mi mayor agradecimiento es para el Profesor Nicolaides, quien desde el primer momento ha compartido conmigo su sueño de “cambiar el mundo”, su conocimiento, pasión y entusiasmo por la medicina fetal, la investigación científica y el trabajo bien hecho. Estos tres años trabajando a tu lado han sido toda una lección de vida y tú has sido mi consejero tanto en mis decisiones profesionales como en las personales. Gracias no sólo por dirigir mi tesis, sino por darme la oportunidad de creer en mi sueño y, lo que es más importante, por darme el coraje y los medios para luchar por conseguirlo.

También le estoy muy agradecida al Profesor Bartha, quien me guió al principio de mi camino en el mundo de la medicina fetal y también en mis primeros pasos hacia la investigación. Gracias por tu apoyo en la distancia durante estos años. Es un honor que hayas dirigido mi tesis junto con el Profesor Nicolaides.

Muchas gracias a Rocío por tantos momentos inolvidables durante nuestras largas horas de trabajo. No sólo has sido la mejor compañera sino que te has convertido en una de mis mejores amigas. Estoy realmente contenta de que Alex, Karen y Konstantinos, fueran también elegidos para desarrollar el estudio conmigo, ya que han hecho un trabajo perfecto. He sido muy afortunada por haber contado con estos cuatro grandes profesionales, a quienes estoy sinceramente agradecida por su contribución.

También quiero agradecer a todos los co-autores de los artículos, en especial a Liona, Ranjit y a Sol, cuyo conocimiento, energía y entusiasmo han sido claves para su desarrollo. Y por supuesto, darle las gracias a todos los fellows que han trabajado para el buen curso de los estudios. Sin vuestro gran esfuerzo ninguno de estos estudios hubiera sido posible. He aprendido mucho de todos.

Le estoy muy agradecida a la Fetal Medicine Foundation, que ha subvencionado todos los estudios y que ayuda cada año a que cientos de médicos como yo, consigan que sus sueños se hagan realidad. Gracias a Ariosa Diagnostics Inc. por analizar las muestras de nuestros estudios. Y gracias a la Fundación Alfonso Martín Escudero, por creer que los jóvenes investigadores españoles todavía tenemos mucho que hacer, y por ayudarnos a que nuestra única preocupación sea hacer un buen trabajo.

Un agradecimiento muy especial es para mi querida familia, quien ha sido una fuente constante de amor, cuidado, apoyo y fortaleza todos estos años. Sé que siempre puedo contar con todos vosotros cuando las cosas se ponen difíciles.

Espero que hoy estéis orgullosos.

Y finalmente, gracias a José, quien ha cambiado mi vida y a quien dedico esta

tesis. No hay palabras que puedan expresar mi gratitud por todo lo que has

hecho. En especial, por creer en mi proyecto y apoyarme, incluso si eso ha

supuesto estar separados más de dos años. Gracias por acompañarme en esta

aventura, ¿cuándo es la próxima?

CONTENTS

CHAPTER 1 INTRODUCTION

1.1 PRESENTACIÓN EN ESPAÑOL

1.2 SCREENING FOR TRISOMIES 21, 18 AND 13 1.2.1 Overview

1.2.2 Prevalence

1.2.3 Evolution in screening from maternal age to the first-trimester combined test 1.2.4 Uptake of invasive test after high-risk result from screening

1.3 SCREENING FOR SEX-CHROMOSOME ANEUPLOIDIES 1.4 SCREENING FOR TRIPLOIDY

1.5 CELL-FREE DNA ANALYSIS IN MATERNAL BLOOD IN SCREENING FOR FETAL TRISOMIES

1.5.1 Overview

1.5.2 Techniques for analysis of cell-free DNA in maternal blood 1.5.3 Importance of fetal fraction

1.5.4 Performance of screening for trisomies

1.5.5 Models for clinical implementation of cfDNA testing

1.6 CONCLUSIONS AND CHALLENGES

1.6.1 Screening for trisomies 21, 18 and 13 in singleton pregnancies 1.6.2 Screening for trisomies 21, 18 and 13 in twin pregnancies

1.6.3 Implementation models for screening for trisomies 21, 18 and 13 in early screening for aneuploidies

1.7 HYPOTHESIS

1.8 OBJECTIVES

CHAPTER 2 PUBLISHED STUDIES

SECTION A: Studies leading to the development of the protocol

STUDY 1 Nicolaides KH, Syngelaki A, Gil M, Atanasova V, Markova D. Validation of targeted sequencing of single-nucleotide polymorphisms for non- invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y. Prenat Diagn 2013; 33:575-579.

STUDY 2 Nicolaides KH, Musci TJ, Struble CA, Syngelaki A, Gil MM. Assessment of fetal sex chromosome aneuploidy using directed cell-free DNA analysis. Fetal Diagn Ther 2014; 35:1-6.

STUDY 3 Nicolaides KH, Syngelaki A, del Mar Gil M, Quezada MS, Zinevich Y.

Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood. Fetal Diagn Ther 2014; 35:212-217.

STUDY 4 Gil MM, Quezada MS, Bregant B, Ferraro M, Nicolaides KH.

Implementation of maternal blood cell-free DNA testing in early screening for aneuploidies. Ultrasound Obstet Gynecol 2013; 42:34-40.

STUDY 5 Bevilacqua E, Gil MM, Nicolaides KH, Ordoñez E, Cirigliano V, Dierickx H, Willems PJ, Jani JC. Performance of screening for aneuploidies by cell-free DNA analysis of maternal blood in twin pregnancies. Ultrasound Obstet Gynecol 2015; 45: 61-66.

SECTION B: Meta-analysis of validation and clinical implementation studies

STUDY 6 Gil MM, Akolekar R, Quezada MS, Bregant B, Nicolaides KH. Analysis of cell-free DNA in maternal blood in screening for aneuploidies: meta- analysis. Fetal Diagn Ther 2014;35:156–173.

STUDY 7 Gil MM, Quezada MS, Revello R, Akolekar R, Nicolaides KH. Analysis of cell-free DNA in maternal blood in screening for fetal aneuploidies:

updated meta-analysis. Ultrasound Obstet Gynecol 2015;45:249-266.

SECTION C: Theoretical models leading to the development of the protocol

STUDY 8 Nicolaides KH, Wright D, Poon LC, Syngelaki A, Gil MM. First-trimester contingent screening for trisomy 21 by biomarkers and maternal blood cell-free DNA testing. Ultrasound Obstet Gynecol 2013;42:41-50.

STUDY 9 Nicolaides KH, Syngelaki A, Poon LC, Gil MM, Wright D. First-trimester contingent screening for trisomies 21, 18 and 13 by biomarkers and maternal blood cell-free DNA testing. Fetal Diagn Ther 2014;35:185-192.

SECTION D: Implementation of the study protocol

STUDY 10 Gil MM, Giunta G, Macalli EA, Poon LC, Nicolaides KH. UK NHS pilot study on cell-free DNA testing in screening for fetal trisomies: factors affecting uptake. Ultrasound Obstet Gynecol 2015;45:67-73.

STUDY 11 Gil MM, Revello R, Poon LC, Akolekar R, Nicolaides KH. Clinical implementation of routine screening for fetal trisomies in the NHS: cell- free DNA test contingent on results from first-trimester combined test.

Ultrasound Obstet Gynecol 2015; in press.

CHAPTER 3 RESULTS SUMMARY (Spanish and English) CHAPTER 4 DISCUSSION

CHAPTER 5 CONCLUSIONS (Spanish and English)

CHAPTER 6 REFERENCES

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CHAPTER 1. INTRODUCTION

1.1 PRESENTACIÓN EN ESPAÑOL

Los defectos cromosómicos son una de las causas mayores de mortalidad y morbilidad perinatal. La cromosomopatía más prevalente es el síndrome de Down (trisomía 21). Su diagnóstico es la indicación más frecuente para realizar un procedimiento invasivo durante el embarazo (amniocentesis o biopsia corial). Estos procedimientos invasivos causan aborto en un 1% de los casos y por lo tanto deben de realizarse en embarazos considerados de alto riesgo tras la realización de test de cribado.

El test de cribado establecido en la práctica clínica, con mayor tasa de detección y menores falsos positivos, se llama test combinado del primer trimestre y engloba parámetros ecográficos como la medida de la translucencia nucal (TN) y parámetros bioquímicos analizados en el suero materno, como la medida de la fracción ß libre de la gonadotrofina coriónica humana (ß-hCG) y la proteína plasmática placentaria A (PAPP-A). Este test combinado tiene una tasa de detección (TD) del 90% para la trisomía 21 y del 95% para las trisomías 18 y 13 con una tasa de falsos positivos (TFP) del 5%.

En 1997, Lo y colaboradores demostraron la presencia de ADN fetal en el plasma

materno. Este descubrimiento ha revolucionado el campo de la medicina fetal abriendo

un gran abanico en el diagnóstico de patología genética fetal. Las primeras aplicaciones

de este test fueron para diagnosticar condiciones fetales no presentes en la madre, tales

como el sexo varón, el Rhesus (Rh) sanguíneo positivo en madres Rh negativas o

enfermedades autosómicas dominantes como el Corea de Hungtinton, en madres

sanas. La evolución de la técnica ha permitido también desarrollar análisis más

complejos que permiten diagnosticar alteraciones cromosómicas como las trisomías. En

los últimos años han aparecido decenas de publicaciones mostrando que mediante el

análisis de ADN libre en sangre materna es posible detectar más del 99% de los casos

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de trisomía 21 y alrededor del 95% de los casos de trisomías 18 y 13 con una TFP menor del 0.5%.

Sin embargo, la tasa de detección teórica de cualquier test de cribado para trisomías no es sinónimo de la tasa de diagnóstico ni de la tasa de interrupción del embarazo en gestaciones afectas, cuando éste se aplica a una población real. En la práctica clínica, muchas gestantes identificadas como alto riesgo mediante el test de cribado no desean una prueba diagnóstica confirmatoria y muchas gestantes a las que se les diagnostica un feto afecto desean continuar con el embarazo.

La hipótesis de los estudios incluidos en esta tesis es que el análisis del ADN libre en sangre materna puede ser usado de rutina en el primer trimestre del embarazo para el cribado de trisomías fetales, tanto en gestaciones únicas como en múltiples.

Para ello, los objetivos de estos estudios fueron, en primer lugar, examinar el rendimiento del test para trisomías 21, 18 y 13, aneuploidías sexuales y triploidías en el primer trimestre del embarazo, mediante estudios de validación e implementación clínica y mediante meta-análisis de la literatura actual. En segundo lugar, explorar la factibilidad de introducir un cribado universal para trisomías 21, 18 y 13 mediante ADN libre a las 10 semanas de gestación conjuntamente con test combinado a las 12.

Tercero, examinar el rendimiento potencial de un modelo de cribado contingente para trisomías 21, 18 y 13, realizando una primera línea de cribado universal mediante el test combinado del primer trimestre y ofreciendo el test de ADN libre a un subgrupo de pacientes identificadas mediante un punto de corte pre-establecido según las TD y TFP que se quieran alcanzar. Y por último, aplicar esta estrategia de cribado contingente en una población de rutina y examinar el rendimiento real de la misma.

Los estudios incluidos en esta tesis, publicados en revistas científicas de alto impacto

en la especialidad, resumen el trabajo realizado por la doctoranda durante su estancia

de formación en el centro de investigación “Harris Birthright Research Center for Fetal

Medicine” en el King´s College Hospital de Londres.

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El aporte de estas investigaciones permitirá implementar el test de AND libre en

poblaciones de rutina, aumentando la TD de las principales trisomías y disminuyendo

de la TFP, con la consecuente disminución en la tasa de realización de procedimientos

invasivos.

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1.2 SCREENING FOR TRISOMIES 21, 18 AND 13

1.2.1 Overview

Chromosomal abnormalities are major causes of perinatal death and childhood handicap. Consequently, diagnosis of chromosomal disorders constitutes the most frequent indication for invasive prenatal diagnosis. However, invasive testing by amniocentesis or chorionic villous sampling is associated with a risk of miscarriage of about 1% and therefore these tests are carried out only in pregnancies considered to be at high-risk for chromosomal defects.

The phenotypic features of Down syndrome were first described in 1866 by Langdon Down.

In 1959 a French pediatrician / geneticist Professor Jerome Lejeune discovered that individuals with Down syndrome have an extra chromosome 21.

The first prenatal diagnosis of trisomy 21 by amniocentesis and fetal karyotyping was in 1968.

The critical factors in a screening test are the ability to discriminate between affected and unaffected individuals and this is expressed in terms of the detection rate and the false positive rate.

Detection rate (or sensitivity)

The detection rate (DR) is the ability of a test to give a positive result in individuals who have the condition being screened for. It is the proportion of affected individuals yielding a positive result (Figure 1.1).

Screen positive and false positive rate

The screen positive rate is the proportion of affected and unaffected individuals yielding

a positive result. The false positive rate (FPR) is the proportion of unaffected

individuals yielding a positive result (Figure 1.1). In screening for chromosomal defects

the term screen positive is usually replaced with false positive because the vast

majority of screen positive cases are actually normal.

INTRODUCTION

21 Figure 1.1 Proportion of affected and unaffected cases above a certain cut-off represent the

detection rate and false positive rate, respectively.

When a screening test has a continuous variable as the result the detection rate and false positive rate vary and are determined by the chosen cut off level (Figure 1.1). The smaller the overlap between affected and unaffected distributions the better the screening test will be. If the frequency distribution is known it is possible to derive the detection rate for a given false positive rate and vice versa by calculating the area under the curves for affected and unaffected cases.

1.2.2 Prevalence

The risk for trisomies increases with maternal age. Additionally, because chromosomally abnormal fetuses are more likely to die in utero than euploid fetuses, the risk decreases with gestational age (Tables 1.1-1.3). The rate of fetal death between 12 weeks (when first trimester screening is performed) and term is about 30%

for trisomy 21 and 80% for trisomies 18 and 13

.

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Table 1.1. Estimated risk for trisomy 21 in relation to maternal age and gestation

Maternal age (years)

Gestational age

10 weeks 12 weeks 14 weeks 16 weeks 20 weeks 40 weeks 20

25 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

1/983 1/870 1/576 1/500 1/424 1/352 1/287 1/229 1/180 1/140 1/108 1/82 1/62 1/47 1/35 1/26 1/20

1/1068 1/946 1/626 1/543 1/461 1/383 1/312 1/249 1/196 1/152 1/117 1/89 1/68 1/51 1/38 1/29 1/21

1/1140 1/1009

1/668 1/580 1/492 1/409 1/333 1/266 1/209 1/163 1/125

1/95 1/72 1/54 1/41 1/30 1/23

1/1200 1/1062

1/703 1/610 1/518 1/430 1/350 1/280 1/220 1/171 1/131 1/100 1/76 1/57 1/43 1/32 1/24

1/1295 1/1147 1/759 1/658 1/559 1/464 1/378 1/302 1/238 1/185 1/142 1/108 1/82 1/62 1/46 1/35 1/26

1/1527 1/1352 1/895 1/776 1/659 1/547 1/446 1/356 1/280 1/218 1/167 1/128 1/97 1/73 1/55 1/41 1/30

Table 1.2. Estimated risk for trisomy 18 in relation to maternal age and gestation

Maternal age (years)

Gestational age

10 weeks 12 weeks 14 weeks 16 weeks 20 weeks 40 weeks 20

25 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

1/1993 1/1765 1/1168 1/1014 1/860 1/715 1/582 1/465 1/366 1/284 1/218 1/167 1/126 1/95 1/71 1/53 1/40

1/2484 1/2200 1/1456 1/1263 1/1072 1/891 1/725 1/580 1/456 1/354 1/272 1/208 1/157 1/118 1/89 1/66 1/50

1/3015 1/2670 1/1766 1/1533 1/1301 1/1081 1/880 1/703 1/553 1/430 1/330 1/252 1/191 1/144 1/108 1/81 1/60

1/3590 1/3179 1/2103 1/1825 1/1549 1/1287 1/1047 1/837 1/659 1/512 1/393 1/300 1/227 1/171 1/128 1/96 1/72

1/4897 1/4336 1/2869 1/2490 1/2490 1/1755 1/1429 1/1142 1/899 1/698 1/537 1/409 1/310 1/233 1/175 1/131 1/98

1/18013 1/15951 1/10554 1/9160 1/7775 1/6458 1/5256 1/4202 1/3307 1/2569 1/1974 1/1505 1/1139 1/858 1/644 1/481 1/359

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23 Table 1.3. Estimated risk for trisomy 13 in relation to maternal age and gestation

Maternal age (years)

Gestational age

10 weeks 12 weeks 14 weeks 16 weeks 20 weeks 40 weeks 20

25 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

1/6347 1/5621 1/3719 1/3228 1/2740 1/2275 1/1852 1/1481 1/1165 1/905 1/696 1/530 1/401 1/302 1/227 1/170 1/127

1/7826 1/6930 1/4585 1/3980 1/3378 1/2806 1/2284 1/1826 1/1437 1/1116 1/858 1/654 1/495 1/373 1/280 1/209 1/156

1/9389 1/8314 1/5501 1/4774 1/4052 1/3366 1/2740 1/2190 1/1724 1/1339 1/1029 1/784 1/594 1/447 1/335 1/251 1/187

1/11042 1/9778 1/6470 1/5615 1/4766 1/3959 1/3222 1/2576 1/2027 1/1575 1/1210 1/922 1/698 1/526 1/395 1/295 1/220

1/14656 1/12978 1/8587 1/7453 1/6326 1/5254 1/4277 1/3419 1/2691 1/2090 1/1606 1/1224 1/927 1/698 1/524 1/392 1/292

1/42423 1/37567 1/24856 1/21573 1/18311 1/15209 1/12380 1/9876 1/7788 1/6050 1/4650 1/3544 1/2683 1/2020 1/1516 1/1134 1/846

1.2.3 Evolution in screening from maternal age to the first-trimester combined test

Trisomy 21

In the last 45 years a series of different methods have been used to identify the pregnancies at high-risk of fetal trisomy 21 that could be offered invasive diagnostic testing.

During the 1970s and the early 1980s, advanced maternal age, defined in most countries as over 35 years, was the method of screening. At that time, about 5% of the pregnant women were >35 years old and this population contained about 30% of the affected pregnancies. Since most of the screen positive group were actually normal the word screen positive became synonymous with false positive. Therefore, in screening by maternal age the DR was 30% and the FPR was 5%. In recent years the trend to delay childbearing has resulted in a significant increase in the number of pregnant women >35 years (20%). If all these women were to undergo invasive testing, the DR would be 50%

and FPR 20%.

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In the late 1980s and early 1990s, it was realised that in pregnancies with fetal trisomy 21 there are altered maternal serum concentrations of various feto-placental products, including increased free ß-human chorionic gonadotropin (ß-hCG) and Inhibin A and decreased alpha fetoprotein (AFP) and unconjugated estriol (uE3).

These biochemical changes were combined with maternal age to develop the double test (ß- hCG and AFP), triple test (ß-hCG, AFP and uE3) and the quad test (ß-hCG, inhibin A, AFP and uE3). Screening by this approach was superior to that of maternal age alone with DR of 50-70% at FPR of 5%.

In the 1990s, aneuploidy screening shifted to the first trimester with the ‘Combined’ test which uses ultrasound measurement of fetal nuchal translucency (NT) together with maternal serum concentration of the placental proteins, ß-hCG and pregnancy- associated plasma protein (PAPP-A).

This combined test has a DR of 90% with FPR of 5%. Research suggests that screening using a combination of NT with other ultrasound markers (nasal bone, tricuspid regurgitation and ductus venosus) and serum biochemistry with ß-hCG, PAPP-A and placental growth factor (PLGF) can have a DR of 97% at FPR of 3%.

However, in most hospitals these additional ultrasound and biochemical markers are not used.

Trisomies 18 and 13

A beneficial consequence of screening for trisomy 21 is the early diagnosis of trisomies 18 and 13, which are the second and third most common chromosomal abnormalities.

At 11-13 weeks, the relative prevalence of trisomies 18 and 13 to trisomy 21 are one to three and one to seven, respectively. All three trisomies are associated with increased maternal age, increased fetal NT and decreased maternal serum PAPP-A, but in trisomy 21 serum ß-hCG is increased whereas in trisomies 18 and 13 this is decreased. In addition, trisomy 13, unlike trisomies 21 and 18, is associated with fetal tachycardia, with the heart rate being above the 95

centile of euploid fetuses in 85%

of fetuses with trisomy 13.

Use of the algorithm for trisomy 21 identifies about 75% of fetuses with trisomies 18

and 13. The combined use of the algorithm for trisomy 21 with specific algorithms for

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trisomies 18 and 13 improves the detection of these aneuploidies to 95% with a small increase in false positive rate by about 0.1%.

Screening in twin pregnancies

In twin pregnancies, effective screening for chromosomal abnormalities is provided by a combination of maternal age and fetal NT thickness.

The performance of screening can be improved by the addition of maternal serum biochemistry, but appropriate adjustments are needed for chorionicity.

In dichorionic twins at 11 to 13 weeks, the levels of maternal serum ß-hCG and PAPP-A are about twice as high as in singleton pregnancies, but in monochorionic twins the levels are lower than in dichorionic twins.

In dichorionic twins, patient-specific risks for trisomy 21 are calculated for each fetus based on maternal age and fetal NT, and the detection rate (75–80%) and false positive rate (5% per fetus or 10% per pregnancy) are similar to those in singleton pregnancies.

In the calculation of risk for trisomies, it has been assumed that in each pregnancy the measurements of NT for CRL between the two fetuses were independent of each other. However, recent evidence indicates that in euploid dichorionic twins, the measurements of NT in each twin pair are correlated and this correlation is not a simple reflection of the common effect of sonographers.

In screening in twins it is therefore necessary to take this correlation into account because it has a substantial impact on the estimated patient-specific risk for trisomies. In dichorionic twins the DR of trisomy 21 from the first-trimester combined test is about 90%, at FPR of 6%, which is only mildly higher than in singleton pregnancies.

First-trimester screening allows the possibility of earlier and therefore safer selective

fetocide in cases where one fetus is euploid and the other is abnormal.

An important

advantage of screening by fetal NT is that when there is discordance for a

chromosomal abnormality, the presence of a sonographically detectable marker helps

to ensure the correct identification of the abnormal twin should the parents choose

selective termination.

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In monochorionic twin pregnancies, the FPR of NT screening is higher than in dichorionic twins, because increased NT in at least one of the fetuses is an early manifestation of twin-to-twin-transfusion syndrome, as well as a marker of chromosomal abnormalities.

In the calculation of risk of trisomy 21, the NT of both fetuses should be measured and the average of the two should be considered.

1.2.4 Uptake of invasive test after high-risk result from screening

The theoretical performance of screening for fetal trisomies is not synonymous with the rate of prenatal diagnosis and termination of affected pregnancies. In practice, many women identified by the screening test as being at high-risk do not want confirmatory diagnostic testing and many women with an affected fetus choose to continue with the pregnancy.

Previous large studies of women identified as being at increased risk for trisomy 21, by the first-trimester combined test or second trimester serum biochemistry, reported that the uptake of invasive testing varied between 53% and 78%.

The studies have also highlighted that the rate of invasive testing increases with increasing estimated risk for trisomies. In a previous study of 30,564 singleton pregnancies undergoing first- trimester combined screening the rate of invasive testing increased exponentially with the estimated risk from less than 1% for those with a risk of <1:10,000 to about 20% for a risk of 1:300-1:500 to more than 85% for a risk of >1:100.

These results demonstrate that pregnant women are able to use sophisticated screening information to make scientifically and ethically rational decisions in favor or against invasive testing.

Similarly, previous studies have also reported that women of Afro-Caribbean racial origin are less likely than Caucasian women to accept prenatal diagnosis for chromosomal abnormalities and this has been attributed to socioeconomic factors and cultural differences in attitudes toward pregnancy, termination and / or raising a disabled child.

Therefore, not all screen-positive women opt to have a diagnostic invasive test and this

decision seems to be influenced by several factors, including those arising from the

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results of the screening test itself but also socio-cultural reasons. It is not certain whether the decision of not having an invasive test performed is due to the fear of the risk of miscarriage associated with the procedure, in which case they would opt for a non-invasive test if available, or their desire to keep an affected baby and therefore the results of invasive or non-invasive test to them are irrelevant.

1.3 SCREENING FOR SEX-CHROMOSOME ANEUPLOIDIES

Prenatal screening for fetal aneuploidies has traditionally focused on trisomy 21 and more recently on trisomies 18 and 13. However, sex chromosome aneuploidies, including Turner syndrome (45,XO), Klinefelter syndrome (47,XXY or 48,XXYY), Triple X syndrome (47,XXX), and 47,XYY, with a combined prevalence of 1:500 are more common than the major trisomies.

Although most cases of sex chromosome aneuploidies are generally mild without intellectual disability, some have a well- established phenotype that can include physical abnormalities, learning delays, and infertility.

It may therefore be desirable by some parents that these conditions could be diagnosed prenatally with the option for pregnancy termination.

However, the traditional methods of screening for trisomies, including maternal age, maternal serum biochemical testing and ultrasound examination of the fetus, are not effective in detecting sex chromosome aneuploidies, except some cases of Turner syndrome presenting with cystic hygromas.

1.4 SCREENING FOR TRIPLOIDY

Triploidy, in which the fetus has three copies of all chromosomes, affects about 1% of

recognized conceptions, but it is highly lethal and it is rarely observed in live births. The

prevalence at 12 weeks’ gestation is about 1 in 2000 and this falls to 1 in 250 000 by

20 weeks.

There are two phenotypes of triploidy, depending on whether the origin

of the extra haploid set is paternal (diandric) or maternal (digynic).

The digynic type

is characterized by a small normal looking placenta, severely growth restricted fetus

with pronounced wasting of the body and sparing of the head, normal fetal NT

thickness and very low serum free ß-hCG and PAPP-A. In the diandric type, the

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placenta is enlarged and partially molar, the fetus is only mildly growth restricted, the fetal NT tends to be high and maternal serum free ß-hCG is about 10 times higher than normal. Diandric triploidy can cause severe maternal complications, including severe early-onset preeclampsia and choriocarcinoma.

First trimester combined screening for trisomies 21, 18 and 13 by fetal NT and serum free ß-hCG and PAPP-A has the beneficial side-effect of detecting about 85% of fetuses with triploidy.

However, with the combined test there is a high false positive rate of about 5% and consequent need for unnecessary invasive diagnostic testing and related miscarriage.

1.5 CELL-FREE DNA ANALYSIS IN MATERNAL BLOOD IN SCREENING FOR FETAL TRISOMIES

1.5.1 Overview

Nucleic acids (DNA and RNA) in plasma were first observed 50 years ago. In the early 1970s increased quantities of DNA were verified in the plasma of cancer.

In the late 1980s and 1990s several groups demonstrated that plasma DNA derived from cancer patients displayed tumour-specific characteristics, including decreased strand stability, Ras and p53 mutations, microsatellite alterations, abnormal promoter hypermethylation of selected genes, mitochondrial DNA mutations and tumour-related viral DNA.

Tumour-specific DNA for a wide range of malignancies has been found:

haematological, colorectal, pancreatic, skin, head-and-neck, lung, breast, kidney, ovarian, nasopharyngeal, liver, bladder, gastric, prostate and cervix. In aggregate, the above data show that tumour-derived DNA in plasma is ubiquitous in affected patients, and likely the result of a common biological process such as apoptosis.

Lo et al. in 1997, based on the previous knowledge that DNA of cancer patients can be detected in plasma, performed a study where he demonstrated the presence of fetal DNA in maternal blood.

This discovery has revolutionized the field of fetal medicine.

They used a rapid-boiling method to extract DNA from plasma and serum. DNA from

INTRODUCTION

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plasma, serum, and nucleated blood cells from 43 pregnant women underwent a sensitive Y-PCR assay to detect circulating male fetal DNA from women bearing male fetuses. Fetus-derived Y sequences were detected in 24 (80%) of the 30 maternal plasma samples, and in 21 (70%) of the 30 maternal serum samples, from women bearing male fetuses. None of the 13 women bearing female fetuses, and none of the 10 non-pregnant control women, had positive results for plasma, serum or nucleated blood cells.

1.5.2 Techniques for analysis of cell-free DNA in maternal blood

Massively parallel sequencing

Many millions of molecules of cfDNA in maternal plasma are sequenced and both the chromosomal origin and quantity of each molecule are determined. In trisomic pregnancies the number of molecules derived from the extra chromosome, as a proportion of all sequenced molecules, is higher than in diploid pregnancies. The ability to detect this difference necessitates that firstly, the number of counts for every chromosome is high, and secondly, the amount of cfDNA in maternal blood that is fetal in origin (fetal fraction) should be at least 4%. In MPSS, molecules from all chromosomes are examined with the potential to identify all aneuploidies. However, since chromosome 21 represents only approximately 1.5% of the human genome, it is necessary to sequence many millions of molecules from the complete genome to ensure sufficient chromosome 21 counts for differentiation between trisomy 21 and euploid pregnancies.

Selective sequencing

In this method selective amplification of specific regions on chromosomes 21, 18, 13, X

and Y is carried out before sequencing analysis. The potential advantage of this

approach is reduced cost because the number of regions that need to be sequenced is

substantially lower than with whole genome sequencing in the detection of the specific

aneuploidies of interest.

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Single nucleotide polymorphism (SNP)-based approaches

An alternative to massively parallel sequencing and selective sequencing is an SNP- based method. In this method, both maternal plasma cfDNA, which contains a mixture of maternal and fetal DNA, and buffy coat DNA, which is maternal in origin, are examined. Targeted amplification and sequencing of about 20,000 polymorphic loci on chromosomes 21, 18, 13, X, and Y is carried out.

A statistical method is then used to analyze allele distributions and determine the chromosomal count of the five chromosomes interrogated in each sample without the need to use a disomic reference chromosome. The method also requires that the minimum fetal fraction is about 4%.

1.5.3 Importance of the fetal fraction

Cell-free DNA in maternal plasma is a mixture of DNA fragments belonging to both the mother and fetus. The proportion of fetal to total cfDNA, referred to as fetal fraction is about 10%. The ability to detect the small increase in the amount of a given chromosome in maternal plasma in a trisomic compared to a disomic pregnancy is directly related to the fetal fraction. For example, if the fetal fraction is 20% in 100 units of maternal cfDNA there would be 20 units coming from the fetus and 80 from the mother; in the presence of a fetus with trisomy 21, the maternal plasma cfDNA would contain 30 units of chromosome 21 coming from the fetus and 80 from the mother.

Thus, there would be 110 units of chromosome 21 compared to 100 units of any other chromosome. This relative increase in chromosome 21 in maternal plasma cfDNA (110 vs. 100) is easier to detect than in a case with a fetal fraction of 4% where the relative increase would be 102 vs. 100.

Current methods of cfDNA testing necessitate that the minimum fetal fraction should be

4%. The greatest risk factor for low fetal fraction is obesity with a small contribution

from Afro-Caribbean racial origin and early gestational age.

The estimated

proportion of pregnancies with fetal fraction below 4% increases with maternal weight

from <1% at 60 kg to >50% at 160 Kg.

The source of fetal cfDNA in maternal plasma

is dying cells in the placenta and the inverse association between fetal fraction and

INTRODUCTION

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maternal weight is likely to be due to a dilutional effect.

A contributing factor is increase in maternal cfDNA levels because with increased weight there is active remodeling of adipose tissue with accelerated turnover of adipocytes.

The fetal fraction increases with increasing serum pregnancy-associated plasma protein-A and free β-human chorionic gonadotropin and is inversely related to maternal weight; the levels are not significantly altered in pregnancies with fetal trisomy 21 but they are reduced in those with trisomy 18.

It is therefore expected that, in trisomies 18 and 13, the failure rate of the cfDNA test would be increased, thereby introducing bias if only the cases with results are included in the calculation of the performance of screening. One study has reported that the rate of failed results was considerably higher in aneuploid than in euploid pregnancies.

1.5.4 Performance of screening for trisomies

Screening in singleton pregnancies

Several studies in the last 4 years have reported the clinical validation and/or implementation of analyzing cfDNA in maternal blood in screening for trisomies 21, 18 and 13 and sex chromosome aneuploidies in singleton pregnancies.

These studies reported cfDNA results in relation to fetal karyotype from invasive testing or clinical outcome. Most of these studies were retrospective, using stored samples from pregnancies with known outcome, or prospective, using mainly samples from high-risk pregnancies undergoing invasive testing. Only five studies reported on the clinical implementation of cfDNA testing in routine screening for trisomies in the general population, showing similar results than those obtained in high-risk populations.

Screening in twin pregnancies

In twin pregnancies, while screening by cfDNA testing is feasible, the performance of

screening may be worse than it is in singletons. In twins, cfDNA testing is more

complex, because the two fetuses could be either monozygotic, and therefore

genetically identical, or dizygotic, in which case only one fetus is likely to have any

aneuploidy identified. There is evidence that, in dizygotic twins, each fetus can

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contribute different amounts of cfDNA into the maternal circulation, and the difference can be nearly two-fold.

It is therefore possible, in a dizygotic twin pregnancy discordant for aneuploidy, for the fetal fraction of the affected fetus to be below the threshold (4%) for successful cfDNA testing. This could lead to an erroneous result of low risk for aneuploidy, with a high contribution from the disomic cotwin resulting in a satisfactory total fetal fraction. To avoid this potential mistake, it was proposed that for cfDNA testing in twin pregnancies, the lower fetal fraction of the two fetuses, rather than the total fetal fraction, should be estimated in the assessment of risk for aneuploidies.

However, such a policy may lead to a higher no-result rate in twins compared to that found in singleton pregnancies. There are a few studies reporting the performance of the cfDNA analysis for screening of aneuploidies in twin pregnancies.

No-result rate from cfDNA testing

One issue with cfDNA testing as a method of screening for aneuploidies is failure to provide a result. There are essentially three reasons for such failure: first, problems with blood collection and transportation of the samples to the laboratory, including inadequate blood volume, hemolysis, incorrect labelling of tubes and delay in arrival to the laboratory; second, low fetal fraction (usually below 4%); and third, assay failure for a variety of reasons, including failed DNA extraction, amplification or sequencing.

This issue has been inconsistently reported among the published studies and, due to its important clinical implications, the no-result rate should be analysed and understood further.

1.5.5 Models for clinical implementation of cfDNA testing

Based on the results of published studies, analysis of cfDNA in maternal blood can detect more that 99% of cases of trisomy 21 for FPR of about 0.1%.

This is far superior to the first trimester combined test, which is the best of the currently available methods of screening.

There are essentially two options in the clinical implementation of cfDNA testing: firstly,

routine screening of the whole population and secondly, contingent screening based on

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the results of first-line screening by another method, preferably the first trimester combined test.

cfDNA testing as a first-line method of screening for all pregnancies

The best approach to implement primary screening for trisomies 21, 18 and 13 by cfDNA testing is to take the maternal blood at 10 weeks’ gestation. The results of the test would then be available at the time of the scheduled first-trimester ultrasound examination, which is ideally performed at 12 weeks. Such approach would retain the advantages of firstly, diagnosis of the major trisomies within the first trimester, and secondly, early diagnosis of major fetal defects and assessment of risk for pregnancy complications.

If cfDNA testing reports a high risk for trisomies 21, 18 or 13 it would be important to confirm or refute the result with invasive testing. In contrast, if cfDNA testing reports a low risk for trisomy 21 or 18 the parents can be reassured that it is highly unlikely that the fetus has one of these aneuploidies. In addition, in those cases where cfDNA testing does not provide a result the parents would still have the option of performing the first-trimester combined test for screening of aneuploidies.

cfDNA testing in the high-risk group from the first-trimester combined test

In this model of clinical implementation of cfDNA testing, first-line screening is by the combined test and in the high-risk group (more than 1:100) cfDNA testing rather than invasive testing is carried out.

If invasive testing was carried out in all cases in the high-risk group, about 90% of

fetuses with trisomy 21 and 95% with trisomies 13 and 18 could be detected at an

overall invasive testing rate of 5%. In this policy of carrying out cfDNA testing in the

high-risk group and reserving invasive testing for the screen positive cases, the overall

invasive testing rate would be substantially reduced to less than 1%. Such strategy,

would be cost neutral, if the cost of cfDNA testing is similar to that of invasive testing,

and would reduce the number of invasive testing related miscarriages. The

disadvantage relates not only to the decrease in the detection of trisomies 21, 18 and

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13 but also the ineffectiveness of cfDNA testing, compared to invasive testing, in detecting other aneuploidies.

cfDNA testing in the intermediate-risk group from the first-trimester combined test

In this model, combined screening is used to divide the population into very a high-risk, intermediate-risk group and low-risk group. In the very high-risk group invasive testing is carried out in all cases. In the intermediate-risk group cfDNA testing is carried out followed by invasive testing for those with a screen positive result. Such strategy could potentially lead to very high DR and very low FPR and consequently, lower invasive test rate.

This approach of cfDNA testing contingent on the results of first-line screening by ultrasound and biochemical testing, retains the major advantages of cfDNA testing in increasing DR and decreasing FPR, but at considerably lower cost than offering the test to the whole population. The approach also retains the benefits achieved by first trimester ultrasound and biochemistry testing in the early detection of major defects and prediction of a wide range of pregnancy complications, which allows for earlier therapeutic intervention and better pregnancy management.

1.6 CONCLUSIONS AND CHALLENGES

1.6.1 Screening for trisomies 21, 18 and 13 in singleton pregnancies

There is extensive evidence that effective screening for trisomies 21, 18 and 13 is provided by a combination of maternal age fetal NT thickness and maternal serum free ß-hCG and PAPP-A at 11-13 weeks’ gestation. The DR is about 90% for trisomy 21 and 95% for trisomies 18 and 13, at combined FPR of 5%.

There is evidence, from clinical validation and a few clinical implementation studies, that

the performance of screening for trisomies by cfDNA analysis of maternal blood is

superior to that of the combined test.

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Additionally, there are few studies, on a limited number of cases and a wide gestational age range, reported on the clinical implementation of cfDNA testing in routine screening for trisomies in the general population.

1.6.2 Screening for trisomies 21, 18 and 13 in twin pregnancies

In twin pregnancies, the performance of screening by the first-trimester combined test for trisomies is similar to that in singleton pregnancies but with a small increase in the FPR. Screening for trisomies in twins by cfDNA analysis of maternal blood is feasible.

However, the number of cases reported is too small to allow accurate estimation of performance of screening.

1.6.3 Implementation models for screening for trisomies 21, 18 and 13 in early screening for aneuploidies

There are essentially two options to implement cfDNA analysis of maternal blood in a general population: firstly, routine screening of the whole population by cfDNA testing and secondly, contingent screening by cfDNA testing on the basis of the results from first-line screening by another method. This method should preferably be the first trimester combined test, since it is the best of the currently available. However, these proposed models need to be implemented in prospective screening to assess their feasibility and performance.

1.7 HYPOTHESIS

Several externally blinded validation studies in the last four years have shown that it is

now possible, through analysis of cell-free cfDNA in maternal blood, to detect more that

99% of cases of trisomy 21, and also a high proportion of those with trisomies 18 and

13 with a FPR of less than 1%. Although most studies were in high-risk pregnancies

there are a few performed in a routine low-risk population, suggesting that cfDNA

testing is applicable not only to pregnancies at high-risk for aneuploidies but also to the

general population where the prevalence of fetal trisomy 21 is much lower.

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The hypothesis of the studies in this thesis is that cfDNA analysis of maternal blood can be used routinely in the first trimester of pregnancy to screen for fetal trisomies, both in singleton and in twin pregnancies. Such policy would lead to higher DR and lower FPR than by the combined test, but would retain the advantages of the combined test, in terms of early confirmation of fetal viability, diagnosis of multiple pregnancy and determination of chorionicity, accurate assessment of gestational age, early detection of major fetal defects and prediction of pregnancy complications, such as preeclampsia.

1.8 OBJECTIVES

The aims of this thesis are:

• To assess the performance of cfDNA analysis in maternal blood for screening of trisomies 21, 18, and 13, sex chromosome aneuploidies and triploidy, both in singleton and twin pregnancies.

• To explore the feasibility of introducing universal screening for trisomies by cfDNA testing at 10 weeks’ gestation and combined testing at 12 weeks.

• To examine the potential performance of screening for trisomies 21, 18 and 13 by a strategy of cfDNA testing in maternal blood contingent on the results of first-line testing by the first trimester combined test, defining risk cut-offs with corresponding DR and FPR.

• To review clinical validation or implementation studies of maternal blood cfDNA testing and define the performance of screening for fetal trisomies 21, 18 and 13 and sex chromosome aneuploidies by a meta-analysis and examine the failure rate in such studies.

• To examine the performance of screening for trisomies 21, 18 and 13 by a strategy of

cfDNA testing in maternal blood contingent on the results of first-line testing by the

first trimester combined test in a routine population.

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CHAPTER 2. PUBLISHED STUDIES

SECTION A: Studies leading to the development of the protocol

STUDY 1 Nicolaides KH, Syngelaki A, Gil M, Atanasova V, Markova D. Validation of targeted sequencing of single-nucleotide polymorphisms for non- invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y. Prenat Diagn 2013; 33:575-579.

STUDY 2 Nicolaides KH, Musci TJ, Struble CA, Syngelaki A, Gil MM. Assessment of fetal sex chromosome aneuploidy using directed cell-free DNA analysis. Fetal Diagn Ther 2014; 35:1-6.

STUDY 3 Nicolaides KH, Syngelaki A, del Mar Gil M, Quezada MS, Zinevich Y.

Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood. Fetal Diagn Ther 2014; 35:212-217.

STUDY 4 Gil MM, Quezada MS, Bregant B, Ferraro M, Nicolaides KH.

Implementation of maternal blood cell-free DNA testing in early screening for aneuploidies. Ultrasound Obstet Gynecol 2013; 42:34-40.

STUDY 5 Bevilacqua E, Gil MM, Nicolaides KH, Ordoñez E, Cirigliano V, Dierickx

H, Willems PJ, Jani JC. Performance of screening for aneuploidies by

cell-free DNA analysis of maternal blood in twin pregnancies. Ultrasound

Obstet Gynecol 2015; 45: 61-66.

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Many studies have shown the feasibility of screening for trisomies with cfDNA analysis of maternal blood. However, none has specifically addressed the issue of undertaking such screening in the first trimester. In most developed countries the introduction of the first trimester combined test has shifted screening and prenatal diagnosis to the first trimester with the advantages of firstly, early reassurance for the majority of parents that their baby is unlikely to be abnormal and secondly, for the few where the baby is abnormal there is an option for earlier and safer pregnancy termination. Therefore, there will be major advantages if the cfDNA test and its subsequent confirmatory invasive tests are completed within the first trimester.

Studies 1-3: These case-control studies were on stored plasma samples from pregnancies with known outcome or prospectively collected maternal samples from high-risk patients before undergoing chorionic villus sampling. They investigate the feasibility of cfDNA testing in screening for trisomies 21, 18 and 13, sex chromosome aneuploidies and triploidy at 11-13 weeks’ gestation.

Study 4: This was a prospective screening study in 1,005 pregnancies where cfDNA testing was carried out at 10 weeks and combined screening at 12 weeks’ gestation.

The study investigates the application of the test in routine screening and examines issues like interval from sampling to obtaining results, failure rate and the need for repeat testing.

Study 5: This was a prospective multicenter screening study in 515 twin pregnancies

where cfDNA testing was carried out at 10-28 weeks’ gestation and the results were

compared to those in 1,847 singleton pregnancies.

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STUDY 1

Nicolaides KH, Syngelaki A, Gil M, Atanasova V, Markova D.

Validation of targeted sequencing of single-nucleotide polymorphisms for non-invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y. Prenat Diagn 2013; 33: 575- 579.

Impact Factor of Prenat Diagn in 2013: 2.514

ORIGINAL ARTICLE

Validation of targeted sequencing of single-nucleotide polymorphisms for non-invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y

K. H. Nicolaides^1,2*, A. Syngelaki¹, M. Gil¹, V. Atanasova¹and D. Markova¹

1Harris Birthright Research Centre for Fetal Medicine, King’s College Hospital, London, UK

2Department of Fetal Medicine, University College London Hospital, London, UK

*Correspondence to: Kypros H. Nicolaides. E-mail: [email protected]

ABSTRACT

Objective To assess the performance of cell-free DNA (cfDNA) testing in maternal blood for detection of fetal aneuploidy of chromosomes 13, 18, 21, X, and Y using targeted sequencing of single-nucleotide polymorphisms.

Methods Prospective study in 242 singleton pregnancies undergoing chorionic villus sampling at 11 to 13 weeks.

Maternal blood was collected before chorionic villus sampling and sent to Natera (San Carlos, CA, USA). cfDNA was isolated from maternal plasma, and targeted multiplex PCR amplification followed by sequencing of 19 488 polymorphic loci covering chromosomes 13, 18, 21, X, and Y was performed. Sequencing data were analyzed using the NATUS algorithm that determines the copy number and calculates a sample-specific accuracy for each of the five chromosomes tested. Laboratory personnel were blinded to fetal karyotype.

Results Results were provided for 229 (94.6%) of the 242 cases. Thirty-two cases were correctly identified as aneuploid, including trisomy 21 [n = 25; sensitivity = 100% (CI: 86.3–100%), specificity = 100% (CI: 98.2–100%)], trisomy 18 (n = 3), trisomy 13 (n = 1), Turner syndrome (n = 2), and triploidy (n = 1), with no false positive or false negative results. Median accuracy was 99.9% (range: 96.0–100%).

Conclusions cfDNA testing in maternal blood using targeted sequencing of polymorphic loci at chromosomes 13, 18, 21, X, and Y holds promise for accurate detection of fetal autosomal trisomies, sex chromosome aneuploidies, and triploidy.

© 2013 John Wiley & Sons, Ltd.

Supporting information may be found in the online version of this article.

Funding sources: The study was supported by a grant from the Fetal Medicine Foundation (UK Charity No: 1037116). Analysis of samples was performed at their own expense by Natera Inc. (San Carlos, CA, USA).

Conflicts of interest: None declared

INTRODUCTION

Prenatal diagnosis of aneuploidies necessitates invasive testing by chorionic villus sampling or amniocentesis in women identified by non-invasive screening to be at increased risk for such aneuploidies. However, invasive testing carries a 1%

risk of causing miscarriage. In the last 40 years, prenatal screening for aneuploidies has focused on trisomy 21, but a beneficial consequence of such screening has been the detection of many additional clinically significant aneuploidies in the screen positive group undergoing invasive testing and full karyotyping. The most effective method of screening for trisomy 21 is by a combination of maternal age, the sonographic measurement of fetal nuchal translucency thickness, and biochemical testing of maternal blood for free b-human chorionic gonadotropin (hCG) and PAPP-A at 11 to 13 weeks’ gestation with a detection rate (DR) of about 90%

and false positive rate (FPR) of 5%.¹ Use of additional

biochemical and sonographic markers, including serum placenta growth factor and assessment of the nasal bone and bloodflow across the tricuspid valve and ductus venosus, can increase the DR to more than 95% and decrease the FPR to less than 3%.¹

Several recent studies have demonstrated that the most effective method of screening for trisomy 21, with DR of more than 99% and FPR of about 0.1%, is derived from examination of cell-free DNA (cfDNA) in maternal plasma.^2–11There are two different approaches in analyzing the cfDNA: quantitative and single-nucleotide polymorphism (SNP)-based methods.

In thefirst approach, maternal plasma cfDNA molecules are sequenced, and the chromosomal origin of each molecule is identified by comparing with the human genome. In trisomic pregnancies, the quantity of molecules derived from the trisomic chromosome, as compared wiith an assumed disomic reference chromosome, is higher than in euploid pregnancies.

Prenatal Diagnosis 2013, 33, 1–5 © 2013 John Wiley & Sons, Ltd.

DOI: 10.1002/pd.4103