Non-invasive prenatal testing (NIPT) for fetal chromosomal aberrations is an important component of healthcare systems worldwide, albeit with varying diagnostic coverage and conditions of use. In Germany, NIPT primarily focuses on trisomies 21, 18 and 13, for which the test costs are reimbursed by the statutory health insurance after thorough prior counseling. Despite this rather restrictive approach compared to other countries, concerns continue to be raised in Germany that young pregnant women, in particular, who are at a low risk of fetal aneuploidy, may have been overly encouraged to undergo NIPT. However, a decision theory-based analysis of the NIPT uptake figures in Germany suggests that there is currently no evidence that avoiding the birth of a trisomic child is a strong motivation particularly of younger women to take the test. Instead, the nation-wide NIPT uptake figures are exceptionally well in line with the corresponding age-specific prior risks. Notably, no such agreement was found when we considered the Netherlands as an example of a healthcare system where NIPT covers additional chromosomal aberrations without age-dependent risk. Replication of our analysis in other countries will reveal whether a strong consistency between age-specific prior risk and NIPT uptake is unique to Germany, or not.

Prenatal non-invasive screening has been offered to pregnant women in Europe since the beginning of this century as first trimester screening (FTS) based on risk calculation programs of Fetal Medicine Foundation (FMF-UK) and FMF-Germany and has spread rapidly worldwide (Nicolaides KH et al. Br J Obstet Gynaecol. 1994;101:782–6, Merz E et al. Ultraschall in Med. 2022;43:115–119). First trimester screening is based on the combination of maternal age, crown rump-length, specific prenatal ultrasound markers such as fetal nuchal translucency (+ optional nasal bone, abnormal ductus venosus flow and tricuspid regurgitation) and the biochemical markers free β-hCG and PAPP-A, and calculates the individual risk for the 3 most common trisomies 13, 18 and 21. Furthermore, the required detailed ultrasound examination allows the detection of structural fetal abnormalities, early growth restriction, twin abnormalities and anomalies of the placenta (Merz E, Pashaj S. Embryonic and early fetal abnormalities diagnosed with three-dimensional ultrasound in the 1st trimester. In: Kurjak A, Chervenak F. Embryo as a person and as a patient. Jaypee Brothers Medical Publishers, New Delhi-London-Panama, 2020; pp. 51–64.2020), leading to a significant improvement in prenatal care beyond trisomies, as aneuploidies account only for 14.26 % of all fetal mal-formations (EUROCAT. https://eu-rd-platform.jrc.ec.europa.eu/eurocat/eurocat-data/preva-lence_en).

In this study we wanted to determine the performance of a paired-end sequencing based noninvasive prenatal testing (NIPT) assay in the detection of common fetal trisomies in twin pregnancy samples. Samples from patients with a twin pregnancy were collected from at least 10 weeks of gestation and analyzed at a single prenatal center in Germany. Results of Anomaly Detected (i.e., high risk) or No Anomaly Detected (i.e., low risk) for trisomy 21, trisomy 18, or trisomy 13 were reported. Followup confirmatory outcomes were requested for all cases. A total of 1,658 patients with twin pregnancies submitted samples during the study period; only two of these samples failed resulting in a low failure rate of 0.12%. Of the remaining 1,656 cases, there were 1,625 (98.1%) low-risk and 31 (1.9%) high-risk NIPTsamples in our cohort. Of these, follow-up information was available for 301 (18.5%) of the low-risk samples and 19 (61.3%) of the high-risk samples. All of the low-risk cases with follow-up were determined to be true negatives giving an estimated negative predictive value of 100%. Seventeen of the 19 high-risk samples with follow-up were true positives, resulting in an overall positive predictive value of 89.5%. Sensitivities of = 99.9% were noted for both trisomy 21 and trisomy 18, with high specificities of = 99.7% observed for all three trisomies. In conclusion, our study showed strong performance of the NIPT assay in the detection of common fetal trisomies in twin pregnancy samples, with high sensitivities, specificities, and positive predictive values observed based on known clinical outcomes along with a low failure rate.

AbstractCombined first-trimester screening (FTS) and noninvasive prenatal testing (NIPT) have been proven to be reliable noninvasive procedures to detect the most common chromosomal abnormalities (trisomies 21, 18, 13) in the first trimester. The aim of this paper is to demonstrate the strengths and limitations of these two procedures and to give a consensus statement of the Fetal Medicine Foundation (FMF) Germany on how to use the two techniques in the first trimester after the introduction of NIPT as a service of the statutory health insurance companies in Germany.

Background: Chromosomal instability, a hallmark of cancer, results in changes in the copy number state. These deviant copy number states can be detected in the cell-free DNA (cfDNA) and provide a quantitative measure of the ctDNA levels by converting cfDNA next-generation sequencing results into a genome-wide copy number instability score (CNI-Score). Our aim was to determine the role of the CNI-Score in detecting epithelial ovarian cancer (EOC) and its role as a marker to monitor the response to treatment. Methods: Blood samples were prospectively collected from 109 patients with high-grade EOC. cfDNA was extracted and analyzed using a clinical-grade assay designed to calculate a genome-wide CNI-Score from low-coverage sequencing data. Stored data from 241 apparently healthy controls were used as a reference set. Results: Comparison of the CNI-Scores of primary EOC patients versus controls yielded sensitivities of 91% at a specificity of 95% to detect OC, respectively. Significantly elevated CNI-Scores were detected in primary (median: 87, IQR: 351) and recurrent (median: 346, IQR: 1891) blood samples. Substantially reduced CNI-Scores were detected after primary debulking surgery. Using a cut-off of 24, a diagnostic sensitivity of 87% for primary and recurrent EOC was determined at a specificity of 95%. CNI-Scores above this threshold were detected in 21/23 primary tumor (91%), 36/42 of platinum-eligible recurrent (85.7%), and 19/22 of non-platinum-eligible recurrent (86.3%) samples, respectively. Conclusion: ctDNA-quantification based on genomic instability determined by the CNI-Score was a biomarker with high diagnostic accuracy in high-grade EOC. The applied assay might be a promising tool for diagnostics and therapy monitoring, as it requires no a priori information about the tumor.

Analysis of 545 NIPT high-risk cases with high risk for trisomy 21 (T21), 18 (T18), and 13 (T13), as well as monosomy X (MX) from routine NIPT testing in a single prenatal center in Germany. Analysis was performed using the VeriSeq NIPT Solution v2 (Illumina Inc., USA). The assessment of true vs false positive results were based on clinical outcome data. The average fetal fraction of 9.7% was within the expected range in T21 and MX but lower in T18 and T13. For all high-risk groups sensitivity and specificity was far above 99%. The positive predictive value (PPV) was highest at trisomy 21 with 94.1%, followed by trisomy 18 with 80.9%. For trisomy 13 and Monosomy X, the PPV was clearly lower at 60.5% and 65.6%, respectively. PPV was dependent on different indications and maternal age. We could show that statistical tools of the method like the log likelihood ratio (LLR) score and T-Statistics value are important to distinguish between (clinical) false positive and true positive NIPT results in trisomies. The relationship between results and quality scores is less significant for MX cases. The study shows that the Illumina VeriSeq v2 procedure is a highly reliable NIPT method with a low no call rate in the hands of experienced diagnostic laboratories.

Noninvasive prenatal testing (NIPT) is a highly sensitive and specific method for detection of fetal chromosomal aneuploidies from maternal plasma. The objective of this study was to determine the performance of a new paired-end sequencing-based NIPT assay in 13,607 pregnancies from a single center in Germany. Samples from 13,607 pregnant women who previously underwent NIPT were analyzed using VeriSeq NIPT Solution v2 assay for presence of common fetal trisomies and monosomy X. Follow-up to determine clinical truth was carried out. Of the 13,607 cases, 13,509 received a NIPT call resulting in a low study failure rate of 0.72%. There were 188 (1.4%) high-risk calls: 117 trisomy 21, 34 trisomy 18, 23 trisomy 13, one trisomy 21 + 13, and 13 monosomy X. High sensitivities and specificities of ≥ 98.89% were reported for all four aneuploidy conditions. Of the high-risk cases, clinical follow-up data were available for 77.1% (145/188). Clinical follow-up of high-risk calls revealed an overall positive predictive value of 84.8% (potential range 65.4–88.3%). NIPT results were provided for samples across a range of fetal fractions, down to 2% fetal fraction. The VeriSeq NIPT Solution v2 assay detected fetal chromosomal aneuploidies across a range of fetal fractions with high sensitivities and specificities observed based on known clinical outcomes, a high overall PPV, and a low failure rate.

ZusammenfassungBei Diagnostik und Monitoring von Tumoren stellten bisher bild- oder biopsiebasierte Verfahren den Goldstandard dar. Neuere Verfahren könnten dieses Vorgehen verändern. So ist es gelungen, zirkulierende Tumorzellen direkt aus dem peripheren Blut zu isolieren und zu charakterisieren. Intratumorale Zellheterogenitäten stellen sich hierbei jedoch als problematisch dar, da diese zu fehlerhaften Betrachtungen führen können. Durch die Analyse von zellfreier Tumor-DNA aus Tumorabbauprodukten im peripheren Blut (sog. Liquid Biopsy) mittels neuester Sequenziertechniken (NGS) können nun auch sehr komplexe Zusammenhänge analysiert werden. Dadurch werden sowohl Einzelgenveränderungen als auch gesamtgenomische Kopienzahlveränderungen (sog. copy number variations, CNV) erkennbar. Bei über 90% der malignen Tumoren lassen sich größere CNV-Abweichungen nachweisen. Der Grad der CNV-Abweichung vom Normalzustand lässt sich in copy number instability (CNI)-Scores standardisieren. Dadurch ist der CNI-Score ein vielversprechender klinischer Biomarker für die Risikostratifizierung und das individualisierte Therapiemonitoring mit dem Potenzial, die Gesundheitskosten und die Krankheitsbelastung für Krebspatienten zu senken.

Background: Screening for fetal trisomy 21 (T21) in the first trimester includes analysis of the serological markers pregnancy-associated plasma protein A (PAPP-A) and free beta-choriogonadotropin (free beta hCG). With the recent launch of these assays on the cobas e and Elecsys platforms, we investigated their clinical and analytical performance.Methods: We conducted a multicenter study in 5397 pregnancies including 108 cross-sectional collected repository cases with verified fetal T21 at 8-14 weeks of gestation. A technical validation of the Roche Elecsys (R) free beta hCG and PAPP-A assays were performed, including method comparisons with the Brahms Kryptor (R), PerkinElmer AutoDEL-FIA (R) and Siemens IMMULITE (R) assays. Furthermore a clinical validation including generation of assay specific medians from gestational age 8+0 to 14+0 weeks, and clinical test performance of risk assessment was performed.Results: The imprecision of the Elecsys free beta hCG and PAPP-A assays was between 1.0% and 2.8%, and both assays showed correlation to Kryptor (free beta hCG 0.981; PAPP-A 0.987), AutoDELFIA (free beta hCG 0.995; PAPP-A 0.979) and IMMULITE assays (free beta hCG 0.983; PAPP-A 0.983). With a cut off at 1: 300 the overall sensitivity of the screening including nuchal translucency reached 94% for a 3% false positive rate.Conclusions: The Roche Elecsys free beta hCG and PAPP-A are suitable and reliable assays for first trimester T21 risk assessment. Both assays were approved and recommended by the FMF.

Background & Patient: Data from 3 008 patients, who underwent single-nucleotide-polymorphism (SNP)-based noninvasive prenatal testing (NIPT) are presented.Method: The PanoramaTM test (Natera, San Carlos, CA) was used to analyze cell-free fetal DNA from maternal blood for trisomies 21, 18, and 13, triploidy and sex-chromosome aneuploidies.Result: In 2 942 (97.8 %) cases, a result was obtained. The average fetal fraction was 10.2 %. A high-risk result for fetal aneuploidy was made for 65 (2.2 %) cases. In 59 (90.8 %) of these cases, invasive testing confirmed the aneuploidy. There were 6 false-positive cases. In the false-positive group, the fetal fraction was significantly lower. The overall positive predictive value was 90.8 %. No false-negative cases were reported but many patients in this study have not delivered yet. Therefore, exact data cannot be given for potential false-negative cases.Conclusion: SNP-based NIPT is a reliable screening method for evaluating the risk of aneuploidies of chromosomes 21, 18 and 13. By using NIPT, the number of invasive procedures may be reduced significantly compared to maternal age and first-trimester screening.

1. cfDNA testing should be offered only after, or in conjunction with, a qualified ultrasound and following appropriate counseling about the nature, scope and significance of the test. 2. cfDNA tests are screening tests. A high-risk cfDNA testing result should always be confirmed by an invasive diagnostic test (Chorionic villous sampling, amniocentesis), before a clinical consequence is drawn from the findings. 3. cfDNA testing can be used as secondary screening test for trisomy 21 (Down syndrome) for the reduction of invasive procedures after a high or intermediate risk result from First-trimester combined test (1 in 1,000 or > 1: 500 (FMF-D)). It should be noted that, even when cfDNA testing is used as a secondary screening, invasive diagnostic testing (Chorionic villous sampling, amniocentesis) is still the method of choice when the adjusted risk for trisomy 21 after the combined test is > 1:10 or the fetal nuchal translucency thickness is > 3.5mm or a fetal malformation is present. 4. cfDNA tests can also be used as a primary screening method for fetal trisomy 21 in pregnant women of every age and risk group. 5. In general, it should be noted that the performance of cfDNA screening for trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome) is lower than that for trisomy 21. 6. Based on the available evidence the use of cfDNA tests to screen for aneuploidy of sex chromosomes and microdeletion syndroms can currently not be recommended without reservation. Correspondence Österreich: Ass. Prof. Priv. Doz. Dr. med. Maximilian Schmid, Universitätsklinik für Frauenheilkunde Wien, E-Mail: maximilian. schmid@meduniwien.ac.at, Assoz. Prof. Priv.-Doz. Dr. Philipp Klaritsch, Universitätsklinik für Frauenheilkunde und Geburtshilfe, Medizinische Universität Graz (Austria), E-Mail: philipp. klaritsch@medunigraz.at Deutschland: Prof. Dr. Bernd Eiben, MVZ Institut für Labormedizin und Klinische Genetik Rhein/Ruhr GmbH, E-Mail: eiben@eurogen.de Schweiz: Prof. Dr. med. Sevgi Tercanli, Ultraschall FreieStrasse Basel, E-Mail: sevgi. tercanli@unibas.ch Recommendation 507

1. cfDNA testing should be offered only after, or in conjunction with, a qualified ultrasound and following appropriate counseling about the nature, scope and significance of the test. 2. cfDNA tests are screening tests. A high-risk cfDNA testing result should always be confirmed by an invasive diagnostic test (Chorionic villous sampling, amniocentesis), before a clinical consequence is drawn from the findings. 3. cfDNA testing can be used as secondary screening test for trisomy 21 (Down syndrome) for the reduction of invasive procedures after a high or intermediate risk result from First-trimester combined test (1 in 1,000 or > 1: 500 (FMF-D)). It should be noted that, even when cfDNA testing is used as a secondary screening, invasive diagnostic testing (Chorionic villous sampling, amniocentesis) is still the method of choice when the adjusted risk for trisomy 21 after the combined test is > 1:10 or the fetal nuchal translucency thickness is > 3.5mm or a fetal malformation is present. 4. cfDNA tests can also be used as a primary screening method for fetal trisomy 21 in pregnant women of every age and risk group. 5. In general, it should be noted that the performance of cfDNA screening for trisomy 18 (Edwards syndrome) and trisomy 13 (Patau syndrome) is lower than that for trisomy 21. 6. Based on the available evidence the use of cfDNA tests to screen for aneuploidy of sex chromosomes and microdeletion syndroms can currently not be recommended without reservation. Correspondence Österreich: Ass. Prof. Priv. Doz. Dr. med. Maximilian Schmid, Universitätsklinik für Frauenheilkunde Wien, E-Mail: maximilian. schmid@meduniwien.ac.at, Assoz. Prof. Priv.-Doz. Dr. Philipp Klaritsch, Universitätsklinik für Frauenheilkunde und Geburtshilfe, Medizinische Universität Graz (Austria), E-Mail: philipp. klaritsch@medunigraz.at Deutschland: Prof. Dr. Bernd Eiben, MVZ Institut für Labormedizin und Klinische Genetik Rhein/Ruhr GmbH, E-Mail: eiben@eurogen.de Schweiz: Prof. Dr. med. Sevgi Tercanli, Ultraschall FreieStrasse Basel, E-Mail: sevgi. tercanli@unibas.ch Recommendation 507

Das Ersttrimesterscreening zur Risikobestimmung für die Trisomien 21, 18 und 13 hat sich in den letzten 15 Jahren in Deutschland etabliert. Die optimale Durchführung setzt die Einhaltung bestimmter Messkriterien beim Ultraschall und bei der biochemischen Analyse voraus sowie die Benutzung evaluierter Risikoberechnungsprogramme wie dem Berechnungsprogramm PRC der Fetal Medicine Foundation Deutschland (FMF-D). Durch die neue Version des Berechnungsprogramms PRC konnten die Trisomie-21-, -18- und -13-Detektionsraten erhöht werden bei gleichzeitiger Senkung der Falsch-positiv-Raten, was einen großen Fortschritt verglichen mit der mütterlichen Altersindikation darstellt.

Zur Früherkennung fetaler Trisomien stehen inzwischen auch Serumtests zur Verfügung, die das Spektrum der nichtinvasiven Diagnostikverfahren erweitern. Welche klinische Bedeutung haben diese Testsysteme, was können sie leisten und wo haben sie auch ihre Grenzen?