The landscape for prenatal diagnostics varies widely across Europe, but in all cases there are dramatic shifts in the range of technology that is available, both for invasively-obtained samples and for non-invasive testing. Cytogenetic arrays and next-gen sequencing are becoming much more common, but there are numerous key issues beyond just technology.  Issues related to access, test interpretation, genetic counseling and implementation of newer tests will all have an impact on changes in the Prenatal Diagnostics landscape in Europe.

Final Agenda

Monday, 13 April

12:00 – 13:00 Registration

Analysis of Invasively-Obtained Samples

13:00 Chairperson’s Opening Remarks

13:05 Current Status, Issues and Challenges for Molecular Analysis of Invasively-Obtained Samples

Marta Rodriguez de Alba, Ph.D., Genetics, Fundacion Jimenez Diaz, Spain

The uptake of different types of molecular analysis for invasively obtained prenatal samples has varied considerably depending on technology and location. QF-PCR rapidly replaced FISH analysis for rapid aneuploidy diagnosis. The transition to Chromosomal Microarray Analysis (CMA) has been slower, but recently has become much more widely accepted. Differences in the incorporation and reimbursement of CMA across countries in Europe will be presented. Some of the challenges for CMA, and approaches for addressing these challenges, will also be discussed.

13:35 Prenatal Diagnosis Using Software-Targeted Array CGH: Robust, Rapid and Unequivocal

Joo Wook Ahn, Ph.D., Guy’s and St. Thomas’ NHS Foundation Trust

Prenatal testing of invasively-obtained samples should demonstrate robust performance and rapid turn-around times and should be affordable within a state-funded health service. Known pathogenic CNVs should be captured, and, ideally, equivocal results should be avoided to minimize parental anxiety and delays in reporting. We have implemented a prenatal array CGH service using software-targeting, designed to detect CNVs of greater than 3Mb, and imbalance in regions of known pathogenicity. All our results are therefore unequivocal and can be reported immediately. As new regions of pathogenicity are published, these can be added to the software-targeting, as can any loci relevant to specific ultrasound anomalies. This service started in March 2012; the results of over 700 samples will be presented and discussed.

14:05 Confirmatory Invasive Testing after a Positive NIPT Result: Chorionic Villus Sampling or Amniocentesis?

Diane Van Opstal, Ph.D., Clinical Genetics, Erasmus Medical Center, The Netherlands

Non-invasive prenatal testing (NIPT) for fetal trisomy detection already revealed that there is a small chance of a false positive and false negative result. This is partly due to the fact that the fetal DNA present in the cell-free maternal plasma fraction is derived from the cytotrophoblast of chorionic villi (CV). From cytogenetic studies in CV we know that the cytotrophoblast is not always representative for the fetus due to chromosomal mosaicism. Therefore, a positive NIPT should always be confirmed with invasive testing in order to investigate the fetal karyotype. The fact that NIPT can be performed from the 10th week of gestation on makes CV sampling, routinely applied between 11-14 weeks of gestation, a more suitable technique for confirmation studies than amniocentesis, mostly carried out after 15,5 gestational weeks. Based on our experience with cytogenetic investigations in CV, the choice for CV sampling or amniocentesis will highly depend on the chromosome aberration involved. For trisomy 13, 18 and 21, we can recommend confirmation studies in CV, provided that these studies include the cytogenetic investigation of both the cytotrophoblast (for confirmation of the NIPT result) and the mesenchymal core (for verification of the fetal karyotype). The protocol for other chromosome aberrations will be shown as well.

14:35 Utilization of a SNP Microarray for High-Resolution Prenatal Studies Over 15,000 Samples

Stuart Schwartz, Ph.D., Strategic Director, Cytogenetics, Laboratory Corporation of America

The findings of over 15,000 prenatal tests utilizing SNP microarray analysis will be reviewed. This data illustrates the utilization not only for ultrasound abnormalities, but also for patients referred for AMA and patients identified with chromosomal anomalies that need better clarification. These studies have not only illustrated the importance of the detection of the gain or loss of chromosomal material, but also the importance of copy-neutral loss of heterozygosity (uniparental disomy or identity by descent). It has also demonstrated the usefulness of examining the SNP alleles for detection of maternal cell contamination, twin-twin contamination, as well as mosaicism and whole genome homozygosity.

15:05 Refreshment Break

Fetal Cell Isolation and Analysis

15:50 Advances in the Isolation and Analysis of Trophoblastic Cells for Non-Invasive Prenatal Diagnosis

Patrizia Paterlini-Brechot, Ph.D., Cellular and Molecular Biology, University of Paris Descartes, France

Trophoblastic cells can be isolated non-invasively from blood and from the cervix at very early terms of pregnancy. The DNA of these fetal cells is not mixed with maternal DNA and can be used efficiently for prenatal, early and non-invasive prenatal diagnosis. Results with consistent isolation and recovery of these cells, and subsequent genetic analysis of DNA from them will be presented. The advantages and remaining challenges for this approach will also be discussed.

16:20 Enrichment of Fetal Nucleated Red Blood Cells from Maternal Blood Using Novel Immunomagnetic Technology (CEPir) for Prenatal Diagnosis

Mahmoud Abuelhija, Ph.D., Research & Development, BioCEP Ltd., Israel

BioCEP has implemented a new method for Fetal Nucleated Red Blood Cells enrichment (fNRBC) from maternal blood, obtained at 8-14 weeks gestation. The new procedure is designed as an alternative to invasive prenatal testing, or to cell-free DNA testing. This approach offers lower risk than invasive approaches, while offering better access to fetal DNA than is achieved with cell-free DNA. Challenges and opportunities for commercialization of this approach in the near future will be discussed.

16:50 ARCEDI - a Rapid Method for Isolation of Fetal Cells from Maternal Blood

Steen Kolvraa, M.D., Chief Scientific Officer, Arcedi Biotech ApS, Denmark

A method for isolation of fetal cells, presumably extravillous trophoblasts, will be presented. The method involves initial PFA fixation of full blood followed by immunomagnetic enrichment of fetal cells using endothelial markers, smearing on slides and identification of fetal cells by ectodermal markers. The total method takes three days. In normal pregnancies we find around ten cells on 30 ml maternal blood.

17:20 Sponsored Presentation (Opportunity Available)

17:50 Close of Day One

TUESDAY, 14 April

10:35 Challenges of Developing a Regulated in vitro Product for NIPT

Stephen Little, Ph.D., CEO, Premaitha Health

Understanding the technical hurdles for launching a quality standard prenatal screening test to given consistency, reliability and robustness in a distrubuted environment. Importance of QA, QC, verification and validation to ensure rapid and reliable uptake.

10:50 NIPT Performance in a Large General Population and Comparison Between High-Risk and Low-Risk Pregnancies

Grover Cong Yu, Ph.D., CEO, BGI-Europe & Africa, BGI Diagnostics

NIPT has been widely used to screen for T21, T18, and T13 in the past few years, yet large clinical data is still absent and concerns have been raised about the performance in large-scale clinical practice. Additionally, validation of NIPT performance in low-risk population is still incomplete. To provide the large clinical data and evaluate the NIPT performance as an audit assessment, we initiated a multi-center prospective study to collect data from the general population in Mainland China from January 1, 2012 to August 31, 2013. To assess the NIPT performance, sensitivity and specificity were calculated with the NIPT positive cases confirmed by karyotyping results, and NIPT negative results confirmed by neonatal physical examination as well as telephone interview at one month after delivery. An insurance program was also used to encourage the report of NIPT false positve and false negative results. Importantly, the study population was divided into high-risk and low-risk groups based on the maternal age, previous Down screening results, ultrasound finding, and pregnancy history. The NIPT performance in the high-risk and low-risk populations was then compared.

11:05 Coffee Break in the Exhibit Hall with Poster Viewing

11:45 Sequencing of Cell-Free DNA as a Marker of Risk for Common Aneuploidies, Pre-Term Labor and Preeclampsia

Leona Poon, MRCOG, M.D., Consultant in Fetal Medicine and Obstetrics, Division of Women’s Health, King’s College London, United Kingdom

Clinical implementation of cell-free DNA testing can either be as routine general screening or as contingent screening, based on other results, such as the first trimester combined test. A comparison of these two approaches, and considerations for interpretation of cell-free DNA results, will be presented. We have also examined the potential for making use of cell-free DNA screening to detect increased risk of preeclampsia or spontaneous preterm labor. Early results and challenges with both of these testing approaches will also be discussed.

12:15 Comparison of Genetic Signatures to Identify Prognostic Markers for the Risk of Pre-Term Birth

Joe Vockley, Ph.D., Chief Scientific Officer, Inova Translational Medical Institute, Inova Fairfax Medical Center, Pediatrics, Virginia Commonwealth University School of Medicine, United States

The Inova Translational Medicine Institute has utilized whole genome sequences and other genomic data from thousands of families to develop a model for predicting the risk of pre-term birth. Data are generated from mother/father/baby trios from a pre-term cohort and a full-term cohort to include families from over 100 countries. RNA markers were found in the peripheral blood of mothers that correlate to pre-term birth but not to common causes of pre-term birth such as premature rupture of membranes and pre-eclampsia. These markers may be critical to the development of a tool for the prediction of preterm birth.

12:45 Luncheon Presentation: Advances in Next-Generation Sequencing for Non-Invasive Prenatal Testing

Alex Helm, MBA, Senior Product Manager, Illumina, Inc.

13:15 Session Break

14:15 Chairperson’s Remarks

14:20 Recovering High Fetal Fractions during Sample Preparation for NIPT: The Potential Role of Point-of-Care Technologies

Maiwenn Kersandy-Kerhoas, Ph.D., School of Engineering and Physical Sciences, Hariot Watt University, United Kingdom

Low fetal fraction of cell-free circulating DNA in the maternal circulation is the primary limitation met by most current prenatal molecular diagnostic technologies and results in sample rejection or lower test reliability. To circumvent this limitation and preserve high fetal fraction, strict sample collection and preparation guidelines have been advocated by clinicians and analysts. Commercial cell-preservation solutions have entered the market, however this area may benefit from a paradigm shift and high fetal fraction preservation and enrichment may lie in on-site sample preparation technologies. A comparative study revealing a 4-fold enrichment of fetal fractions after on-chip maternal blood plasma extraction compared to conventional centrifugation will be presented.

14:50 Clinical Experience Using Targeted Microarrays for NIPT

Tuba Gunel, Ph.D., Molecular Biology and Genetics, Istanbul University, Turkey

Analysis of maternal blood plasma cell-free fetal (cff) DNA using array-CGH allows for detection of whole chromosome differences between test and reference DNA. Such arrays are commonly used to analyze amniotic samples and pre-implantation embryos. We hypothesized that extraction, fluorescent labeling, hybridization, and analysis of cffDNA could be used to simultaneously screen for aneuploidy across every chromosome. This technology may also aid the identification of minor genetic aberrations, such as micro-deletions and micro-duplications, which could enhance prenatal genetic diagnostics. NIPT using microarrays provided faster and more accurate cell-free DNA (cfDNA) measurements than sequencing.

15:20 ANGELAB, Developing Lab-on-a-Chip-Based Systems for Epigenetic Non-Invasive Prenatal Diagnosis

Jesus M. Ruano-Lopez, Ph.D., IK4-Ikerlan, Spain

The European Project called ANGELAB, which is developing a family of In Vitro Diagnostic Systems, will be presented. Each LabonaChip runs fetal DNA sample preparation in combination with real-time or Digital PCR. The methodology and system architecture used to transfer epigenetic biological protocols into modules, then into LabonaChip, and then into integrated systems will be described. The strategy, challenges and progress made over the first two years of this project will also be covered.

15:50 Refreshment Break in the Exhibit Hall with Poster Viewing

18:00 Welcome Reception in the Exhibit Hall with Poster Viewing

19:00 Close of Day Two

WEDNESDAY, 15 April

8:00 Registration and Morning Coffee

NIPT

8:40 Chairperson’s Remarks

8:45 NIPD for Monogenic Disorders without Using Next-Generation Sequencing

Jessica van den Oever, MSc, Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA); Department of Clinical Genetics, Leiden University Medical Center

Next generation sequencing (NGS) is the main method used for non-invasive prenatal testing and diagnosis. However this method is still quite costly and may not be accessible to all laboratories. Moreover, although the approach is very successful for detection of fetal aneuploidies it is also has its limitations. Noninvasive detection of fetal repeat expansions or paternally inherited mutations in certain regions of the genome is currently difficult, if not impossible, by using NGS. A sensitive, fast and low cost alternative for NGS-based targeted mutation scanning will be presented, as well as a validation study for detection of polymorphic paternally inherited CAG repeats for fetuses at risk of Huntington Disease.

9:15 Development of Quantitative PCR for NIPD of Single Gene Disorders

Claire Guissart, Ph.D., Laboratory of Genetic Medicine, University of Montpellier, France

In recent years, non-invasive prenatal diagnosis (NIPD) has found new applications in monogenic disease diagnostics for paternally inherited mutations. This presentation discusses the application of a qPCR-based mutant enrichment technique for NIPD of single gene disorder using Cystic Fibrosis as a clinical application model. In this respect, we have also repurposed a commercial mini-STR kit — frequently used in forensic STR analysis — as an external quality control to confirm the presence of cell-free fetal DNA.

9:45 Experience with over 5000 NIPT Samples: Overcoming Technical and Biological Challenges

Joris R. Vermeesch, Ph.D., Professor, Molecular Cytogenetics and Genome Research, University of Leuven

NIPT for fetal aneuploidy detection is increasingly being offered in the clinical setting. We present an analysis pipeline that enables the differentiation of fetal trisomies from local maternal CNVs and the detection of non-classical aneuploidies as well as segmental imbalances. We present the results from its clinical application on over 4000 prospective pregnancies. Interestingly, our analysis pipeline also identified a pregnant woman with early-stage nodular sclerosis Hodgkin lymphoma.

10:15 Coffee Break in the Exhibit Hall with Poster Viewing

10:45 The Diagnostic Accuracy of Non-Invasive Fetal RHD Typing

Florentine Thurik, M.D., Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands

In the Netherlands non-invasive fetal RHD screening was implemented in July 2011. The evaluation of this screening program shows the high diagnostic accuracy of this approach; only 9 false negative results on > 26.000 serologically confirmed tests. Since the fetal RHD screening is based on a quantitative RHD PCR, we now have access to a huge data set on quantitative fetal DNA concentrations, showing the large biological variability. The causes of false positivity and negativity will be discussed, and the lessons to be learned from this analysis for other non-invasive tests.

11:45 Close of Conference