Neeta L. Vora

Noninvasive prenatal testing and incidental detection of occult maternal malignancies

IMPORTANCE Understanding the relationship between aneuploidy detection on noninvasive prenatal testing (NIPT) and occult maternal malignancies may explain results that are discordant with the fetal karyotype and improve maternal clinical care. OBJECTIVE To evaluate massively parallel sequencing data for patterns of copy-number variations that might prospectively identify occult maternal malignancies. DESIGN, SETTING, AND PARTICIPANTS Case series identified from 125,426 samples submitted between February 15, 2012, and September 30, 2014, from asymptomatic pregnant women who underwent plasma cell-free DNA sequencing for clinical prenatal aneuploidy screening. Analyses were conducted in a clinical laboratory that performs DNA sequencing. Among the clinical samples, abnormal results were detected in 3757 (3%); these were reported to the ordering physician with recommendations for further evaluation. EXPOSURES NIPT for fetal aneuploidy screening (chromosomes 13, 18, 21, X, and Y). MAIN OUTCOMES AND MEASURES Detailed genome-wide bioinformatics analysis was performed on available sequencing data from 8 of 10 women with known cancers. Genome-wide copy-number changes in the original NIPT samples and in subsequent serial samples from individual patients when available are reported. Copy-number changes detected in NIPT sequencing data in the known cancer cases were compared with the types of aneuploidies detected in the overall cohort. RESULTS From a cohort of 125,426 NIPT results, 3757 (3%) were positive for 1 or more aneuploidies involving chromosomes 13, 18, 21, X, or Y. From this set of 3757 samples, 10 cases of maternal cancer were identified. Detailed clinical and sequencing data were obtained in 8. Maternal cancers most frequently occurred with the rare NIPT finding of more than 1 aneuploidy detected (7 known cancers among 39 cases of multiple aneuploidies by NIPT, 18% [95% CI, 7.5%-33.5%]). All 8 cases that underwent further bioinformatics analysis showed unique patterns of nonspecific copy-number gains and losses across multiple chromosomes. In 1 case, blood was sampled after completion of treatment for colorectal cancer and the abnormal pattern was no longer evident. CONCLUSIONS AND RELEVANCE In this preliminary study, a small number of cases of occult malignancy were subsequently diagnosed among pregnant women whose noninvasive prenatal testing results showed discordance with the fetal karyotype. The clinical importance of these findings will require further research.

Discordant noninvasive prenatal testing results in a patient subsequently diagnosed with metastatic disease

University of North Carolina at Chapel Hill School of Medicine, Department of Obstetrics and Gynecology, Division of Maternal Fetal Medicine, Chapel Hill, NC 27599, USA Verinata Health, Inc., Department of Clinical Affairs, Redwood City, CA, USA University of North Carolina at Chapel Hill School of Medicine, Department of Pathology and Laboratory Medicine, Chapel Hill, NC, USA *Correspondence to: Neeta L. Vora. E-mail: nvora@med.unc.edu

Noninvasive prenatal testing for microdeletion syndromes and expanded trisomies: proceed with caution.

The identification of circulating cell-free fetal DNA in maternal plasma has led to the introduction of noninvasive prenatal tests with high sensitivity and high specificity for common aneuploidies (trisomy 13, trisomy 18, trisomy 21). A new expanded noninvasive prenatal testing panel that includes five microdeletion syndromes (22q11 deletion syndrome, cri-du-chat [5p minus], Prader Willi or Angelman syndrome, 1p36 deletion syndrome) and two aneuploidies usually associated with nonviable pregnancies (trisomy 16 and trisomy 22) is now available. This expanded panel will be performed unless an opt-out box is checked. Because these disorders are so rare, the positive predictive value is expected to be low. As with all new screening tests and technologies, the expanded panel should be appropriately studied before it is widely adopted.

Increased Death of Adipose Cells, a Path to Release Cell‐Free DNA Into Systemic Circulation of Obese Women

Remodeling of adipose tissue is required to support the expansion of adipose mass. In obesity, an increased death of adipocytes contributes to the accelerated cellular turnover. We have shown that obesity in pregnancy is associated with metabolic and immune alterations in the adipose tissue. In this study, we characterized the mechanisms responsible for increased death of adipose cells of pregnant obese women and its functional consequences. We postulated that a higher turnover of dead cells in white adipose tissue of obese women would translate into release of cell‐free DNA (cfDNA) into their systemic circulation. Increase in adipose mass of obese compared to lean women results from a lesser number of hypertrophic adipocytes and an accumulation of macrophages in the stromal vascular fraction (SVF). The adipocytes of obese displayed enhanced necrosis with a loss of perilipin staining at the plasma membrane. Apoptosis was prominent in SVF cells with an increased expression of caspase 9 and caspase 3 and a higher rate of terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end‐labeling (TUNEL) positive CD68 macrophages in obese vs. lean. Whereas circulating fetal cfDNA concentrations were not changed, there was a twofold increase in circulating glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) cfDNA and adipose tissue GAPDH mRNA in obese women. The maternal systemic GAPDH cfDNA was positively correlated with BMI and gestational weight gain. These data suggest that the active remodeling of adipose tissue of obese pregnant women results in an increased release of cfDNA of maternal origin into the circulation.

Reproductive Issues for Adults With Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common disorder. However, the consequences of ADPKD on male and female reproductive health are not widely known. Several abnormalities are found in men with ADPKD, including necrospermia, immotile sperm, seminal vesicle cysts, and ejaculatory duct cysts. Female fertility is not affected. Affected women with ADPKD and normal renal function have a high rate of successful uncomplicated pregnancies. Pregnant women with ADPKD with compromised kidney function should be monitored carefully for the development of hypertension and preeclampsia. Their fetuses should be examined sonographically for signs of uteroplacental insufficiency, such as intrauterine growth restriction and oligohydramnios. The diagnosis of ADPKD should always be considered when prenatal sonographic findings of hyperechogenic enlarged kidneys are found. In this setting, a family history and renal sonogram of both parents is indicated. Sequencing of the PKD1 and PKD2 genes is available and can be used for both prenatal and preimplantation genetic diagnosis. We review in detail these topics to familiarize physicians taking care of patients with ADPKD with the reproductive issues that confront affected individuals.

Genetic considerations in the prenatal diagnosis of overgrowth syndromes.

Large (>90%) for gestational age (LGA) fetuses are usually identified incidentally. Detection of the LGA fetus should first prompt the provider to rule out incorrect dates and maternal diabetes. Once this is done, consideration should be given to certain overgrowth syndromes, especially if anomalies are present. The overgrowth syndromes have significant clinical and molecular overlap, and are associated with developmental delay, tumors, and other anomalies. Although genetic causes of overgrowth are considered postnatally, they are infrequently diagnosed prenatally. Here, we review prenatal sonographic findings in fetal overgrowth syndromes, including Pallister‐Killian, Beckwith‐Wiedemann, Sotos, Perlman, and Simpson‐Golabi‐Behmel. We also discuss prenatal diagnosis options and recurrence risks. Copyright

A multifactorial relationship exists between total circulating cell-free DNA levels and maternal BMI

Maternal obesity affects 1 in 5 pregnant women in the United States1. Maternal obesity is associated with increased circulating total, but not fetal, cf DNA2. This may be a result of increased production or decreased clearance of cf DNA in obese women. It is more likely that this is due to increased production of total cfDNA, because decreased clearance would also likely lead to an increase in cell-free fetal cfDNA. In a prior study performed on obese pregnant women, we showed that active remodeling of adipose tissue via adipocyte necrosis and/or apoptosis of the stromal vascular fraction results in an increased release of cfDNA of maternal origin into the circulation3. The focus of the prior study was on the mechanisms underlying the release of the cfDNA. In this study we more closely examined the correlation between maternal weight and total DNA levels. This study was approved by the Institutional Review Boards at Tufts Medical Center and Metrohealth Medical Center. The samples are from the same cohort reported previously3, but the analysis is different. Briefly, sixteen obese (mean=39.2; pre-gravid BMI range 31–51) and 14 lean (mean 21.8; pre-gravid BMI range 17–24) women carrying male fetuses and 10 women carrying female fetuses (negative controls) were recruited at term (37–40 weeks) prior to an elective cesarean section. Written informed consent was obtained prior to obtaining the samples. Women with a multiple gestations, placenta previa or invasive placentation, labor, infection, fetal anomalies or aneuploidy, intrauterine growth restriction, or preeclampsia were excluded. Maternal peripheral venous blood was collected at MetroHealth Medical Center on admission to labor and delivery, prior to placement of an intravenous line for hydration. All subjects had the same instructions prior to admission and had nothing to eat or drink for 6–8 hours prior to the blood draw. The blood was collected in an EDTA tube and plasma was separated by centrifugation and kept frozen at −20°C prior to being shipped to Tufts Medical Center for further analysis. DNA was extracted from 400 uL of plasma using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA) according to the blood and body fluid protocol. DNA was eluted in 50 μL of the elution buffer. Real time quantitative PCR amplification was performed as previously described4 to amplify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS14 as markers of total and fetal DNA, respectively. All samples were analyzed in triplicate. Analysis was blinded, and a female processed and handled all samples so that there was no risk of contaminating samples with male DNA. Conversion of raw PCR data to genome equivalents per mL of plasma was performed using the methods of Lo et al.5 The levels of total and fetal cf DNA in plasma from lean and obese women were compared using the t test. In addition, the maternal plasma volume from both obese and lean subjects was adjusted for blood volume6 and compared using the t test, as prior experiments in our laboratory suggested an increased blood volume as a function of weight7. Finally, we performed a regression analysis between total and fetal cf DNA and maternal BMI. Analysis of the raw data from all subjects showed that there was a 1.7% increase in total cell-free DNA per BMI unit (kg/m2). Following adjustment for blood volume, there was a 3.2% increase per BMI unit (Table 1). When the subjects were categorized into lean and obese groups, however, there was a decrease in total cf DNA per BMI unit in lean women and an increase in obese women (Table 1 and Figure 1). Neither of these changes were statistically significant. However, this lack of significance may be a function of the small sample size. Further studies with more subjects are warranted to further characterize this relationship between cf DNA and BMI. Figure 1 Graph showing relationship between total cell-free DNA levels and body mass index [BMI]. The correlation coefficient between BMI and cfDNA for the lean population is -0.061 (p=0.41) and for the obese population is 0.23 (p=0.20). Table 1 Change in maternal cell-free DNA by body mass index In this study, we found a correlation between BMI and total DNA levels. In lean women, the decrease in total cf DNA likely reflects a dilutional effect seen in all pregnant women due to increasing plasma volume. Conversely, in obese women, the increased levels of total cf DNA may reflect the increased adipocyte necrosis3 and stromal vascular apoptosis that is significant enough to overcome the dilutional effect that occurs in pregnant women. Our results are similar to a previous study by Lapaire et al.2, in which a correlation was found between maternal BMI and total, but not fetal, second trimester cf DNA levels. There are several differences between our study and the Lapaire study, however. Firstly, the gestational ages of the study subjects were different. In the present study, maternal blood samples were drawn at term, in contrast to 20 to 21 weeks. Secondly, in the Lapaire et al. study there was no adjustment for maternal weight. We adjusted for the effect of maternal weight on blood volume using the method described by Lemmens et al.6 The present study and that of Wataganara et al.7 suggest that a correction factor for maternal weight is needed when cf DNA analyses are performed. It is standard practice to correct for maternal weight when performing serum screening for Down syndrome. Diagnostic laboratories typically derive their own regression curves for the relationship between serum analytes and maternal weight because of several factors, including differences in instrument sensitivity in the measurement of serum analytes, as well as differences in maternal weight distribution among centers8. Review of the literature, however, indicates that there is no consensus as to the best method to adjust serum markers, including cf nucleic acids, for maternal weight in cases of extreme obesity. We selected the Lemmens et al.6 formula because it correlated total blood volume with BMI, although that formula was used for quantification of blood volume in non-pregnant surgical patients. In summary, we show here that maternal BMI affects total cf DNA levels in both lean and obese pregnant women. In the future, raw DNA values may need to be adjusted for maternal BMI for adequate interpretation of clinical tests that involve assessment of the fetal fraction9. In addition, the presence of increased total cf DNA levels in obese pregnant women suggests that this analyte may be a biomarker for systemic problems during gestation that may impact both maternal and fetal health.

Maternal Obesity Affects Fetal Neurodevelopmental and Metabolic Gene Expression: A Pilot Study

Objective One in three pregnant women in the United States is obese. Their offspring are at increased risk for neurodevelopmental and metabolic morbidity. Underlying molecular mechanisms are poorly understood. We performed a global gene expression analysis of mid-trimester amniotic fluid cell-free fetal RNA in obese versus lean pregnant women. Methods This prospective pilot study included eight obese (BMI≥30) and eight lean (BMI<25) women undergoing clinically indicated mid-trimester genetic amniocentesis. Subjects were matched for gestational age and fetal sex. Fetuses with abnormal karyotype or structural anomalies were excluded. Cell-free fetal RNA was extracted from amniotic fluid and hybridized to whole genome expression arrays. Genes significantly differentially regulated in 8/8 obese-lean pairs were identified using paired t-tests with the Benjamini-Hochberg correction (false discovery rate of <0.05). Biological interpretation was performed with Ingenuity Pathway Analysis and the BioGPS gene expression atlas. Results In fetuses of obese pregnant women, 205 genes were significantly differentially regulated. Apolipoprotein D, a gene highly expressed in the central nervous system and integral to lipid regulation, was the most up-regulated gene (9-fold). Apoptotic cell death was significantly down-regulated, particularly within nervous system pathways involving the cerebral cortex. Activation of the transcriptional regulators estrogen receptor, FOS, and STAT3 was predicted in fetuses of obese women, suggesting a pro-estrogenic, pro-inflammatory milieu. Conclusion Maternal obesity affects fetal neurodevelopmental and metabolic gene expression as early as the second trimester. These findings may have implications for postnatal neurodevelopmental and metabolic abnormalities described in the offspring of obese women.

A single center’s experience with noninvasive prenatal testing

Purpose:Massively parallel sequencing to detect fetal aneuploidy has high sensitivity and specificity for the detection of trisomies 21, 18, and 13 in high-risk populations. The purpose of our study was to review our institution’s experience with the use of noninvasive prenatal testing for aneuploidy screening.Methods:This was a descriptive study of patients who had undergone noninvasive prenatal testing between January and September 2012 at the UNC Prenatal Diagnosis unit.Results:Two hundred and eight women had undergone noninvasive prenatal testing during the study period. The majority of patients were white (62.9%) and of advanced maternal age (71.2%). The fetal fraction was below the threshold in three obese patients (1.4%). An abnormal noninvasive prenatal test (aneuploidy detected or “unclassified” result) was reported in 6.3% (13/208) of the patients. Noninvasive prenatal testing had a combined sensitivity of 87.5% and specificity of 99.5% for detection of trisomies 21, 18, and 13. There were “unclassified” results in 11.1% (5/45) of the patients. Over the study period, the number of patients requesting noninvasive prenatal testing increased monthly. The rate of amniocenteses significantly declined (8.1% before vs. 5.3% after noninvasive prenatal testing, P < 0.01).Conclusion:An increase in uptake of noninvasive prenatal testing and a significant decline in amniocentesis procedures were observed. The rates of “unclassified,” false-positive, and false-negative results were higher than anticipated based on published preclinical trials.Genet Med 16 9, 681–687.

Promises, pitfalls and practicalities of prenatal whole exome sequencing

Prenatal genetic diagnosis provides information for pregnancy and perinatal decision‐making and management. In several small series, prenatal whole exome sequencing (WES) approaches have identified genetic diagnoses when conventional tests (karyotype and microarray) were not diagnostic. Here, we review published prenatal WES studies and recent conference abstracts. Thirty‐one studies were identified, with diagnostic rates in series of five or more fetuses varying between 6.2% and 80%. Differences in inclusion criteria and trio versus singleton approaches to sequencing largely account for the wide range of diagnostic rates. The data suggest that diagnostic yields will be greater in fetuses with multiple anomalies or in cases preselected following genetic review.