Stephanie Kareht

We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.

BACKGROUND: The 22q11.2 deletion syndrome is the most common microdeletion syndrome in livebirths, but data regarding its incidence in other populations is limited and also include ascertainment bias. This study was designed to determine the incidence of the 22q11.2 deletion in miscarriage samples sent for clinical molecular cytogenetic testing. RESULTS: Twenty-six thousand one hundred one fresh product of conception (POC) samples were sent to a CLIA- certified, CAP-accredited laboratory from April 2010--May 2016 for molecular cytogenetic miscarriage testing using a single-nucleotide polymorphism (SNP)-based microarray platform. A retrospective review determined the incidence of the 22q11.2 deletion in this sample set. Fetal results were obtained in 22,451 (86%) cases, of which, 15 (0.07%) had a microdeletion in the 22q11.2 region (incidence, 1/1497). Of those, 12 (80%) cases were found in samples that were normal at the resolution of traditional karyotyping (i.e., had no chromosome abnormalities above 10 Mb in size) and three (20%) cases had additional findings (Trisomy 15, Trisomy 16, XXY). Ten (67%) cases with a 22q11.2 deletion had the common ~3 Mb deletion; the remaining 5 cases had deletions ranging in size from 0.65 to 1.5 Mb. A majority (12/15) of cases had a deletion on the maternally inherited chromosome. No significant relationship between maternal age and presence of a fetal 22q11.2 deletion was observed. CONCLUSIONS: The observed incidence of 1/1497 for the 22q11.2 deletion in miscarriage samples is higher than the reported general population prevalence (1/4000-1/6000). Further research is needed to determine whether the 22q11.2 deletion is a causal factor for miscarriage.

OBJECTIVE: To identify pregnancies at increased risk for trisomy 13, trisomy 18 or triploidy attributable to low fetal fraction (FF). METHODS: A FF-based risk (FFBR) model was built using data from more than 165 000 singleton pregnancies referred for single-nucleotide polymorphism (SNP)-based non-invasive prenatal testing (NIPT). Based on maternal weight and gestational age (GA), FF distributions for normal, trisomy 13, trisomy 18 and triploid pregnancies were constructed and used to adjust prior risks for these abnormalities. A risk cut-off of ≥ 1% was chosen to define pregnancies at high risk for trisomy 13, trisomy 18 or triploidy (high FFBR score). The model was evaluated on an independent blinded set of pregnancies for which SNP-based NIPT did not return a result, and for which pregnancy outcome information was gathered retrospectively. RESULTS: The evaluation cohort comprised 1148 cases, of which approximately half received a high FFBR score. Compared with rates expected based on maternal age (MA) and GA, cases with a high FFBR score had a significantly increased rate of trisomy 13, trisomy 18 or triploidy combined (5.7% vs 0.7%; P < 0.001) and also of unexplained pregnancy loss (14.7% vs 10.4%; P < 0.001). For cases that did not receive a high FFBR score, the incidence of a chromosomal abnormality or pregnancy loss was not significantly different from that expected based on MA and GA. In this study cohort, the sensitivity of the FFBR model for detection of trisomy 13, trisomy 18 or triploidy was 91.4% (95% CI, 76.9-98.2%) with a positive predictive value of 5.7% (32/564; 95% CI, 3.9-7.9%). CONCLUSIONS: For pregnancies with a FF too low to receive a result on standard NIPT, the FFBR algorithm identified a subset of cases at increased risk for trisomy 13, trisomy 18 or triploidy. For the remainder of cases, the risk of a fetal chromosomal abnormality was unchanged from that expected based on MA and GA. © 2018 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.