Genome-wide cell-free DNA screening: a focus on copy-number variants.

PURPOSE: Of 86,902 prenatal genome-wide cell-free DNA (cfDNA) screening tests, 4,121 were positive for a chromosome abnormality. This study examines 490 cases screen-positive for one or more subchromosomal copy-number variants (CNV) from genome-wide cfDNA screening. METHODS: Cases positive for one or more subchromosomal CNV from genome-wide cfDNA screening and diagnostic outcomes were compiled. Diagnostic testing trends were analyzed, positive predictive values (PPVs) were calculated, and the type of chromosomal abnormalities ultimately confirmed by diagnostic testing were described. RESULTS: CNVs were identified in 0.56% of screened specimens. Of the 490 cases screen-positive for one or more CNV, diagnostic outcomes were available for 244 cases (50%). The overall PPV among the cases with diagnostic outcomes was 74.2% (95% CI: 68.1-79.5%) and 71.8% (95% CI: 65.5-77.4%) for "fetal-only" events. Overall, isolated CNVs showed a lower PPV of 61.0% (95% CI: 52.5-68.8%) compared to complex CNVs at 93.9% (95% CI: 86.6-97.5%). Isolated deletions/duplications and unbalanced structural rearrangements were the most common diagnostic outcomes when isolated and complex CNVs were identified by cfDNA screening, respectively. CONCLUSION: Genome-wide cfDNA screening identifies chromosomal abnormalities beyond the scope of traditional cfDNA screening, and the overall PPV associated with subchromosomal CNVs in cases with diagnostic outcomes was >70%.

Three years of clinical experience with a genome-wide cfDNA screening test for aneuploidies and copy-number variants.

PURPOSE: Pregnant women have unprecedented choices for prenatal screening and testing. Cell-free DNA (cfDNA) offers the option to screen for aneuploidy of all chromosomes and genome-wide copy-number variants (CNVs), expanding screening beyond the common trisomies ("traditional" cfDNA). We sought to review the utilization trends and clinical performance characteristics of a commercially available genome-wide cfDNA test, with a subset having available diagnostic testing outcomes. METHODS: Retrospective analysis of 55,517 samples submitted for genome-wide cfDNA screening at a commercial laboratory, assessing indications, demographics, results, and performance. The cohort was broken into three "testing years"' to compare trends. RESULTS: Indications shifted over time, with a decrease in referrals for ultrasound findings (22.0% to 12.0%) and an increase in no known high-risk indication (3.0% to 16.6%). Of the positive results, 25% would be missed with traditional cfDNA screening. High sensitivity and specificity were observed with a positive predictive value (PPV) of 72.6% for genome-wide CNVs and 22.4% for rare autosomal trisomies (RATs). CONCLUSION: A broader patient population is utilizing genome-wide cfDNA, yet positivity rates and the contribution of genome-wide events have remained stable at approximately 5% and 25%, respectively. Test performance in a real-world clinical population shows high PPVs in those CNVs tested, with diagnostic outcomes in over 40% of positive cases.

Obesity and no call results: optimal timing of cell-free DNA testing and redraw.

BACKGROUND: Fetal fraction of cell-free DNA decreases with increasing maternal weight. Consequently, cell-free DNA screening for fetal aneuploidy has higher screen failures or "no call" rates in women with obesity owing to a low fetal fraction. The optimal timing of testing based on maternal weight is unknown. OBJECTIVE: This study aimed to identify the optimal timing of initial cell-free DNA testing based on maternal weight and to identify the optimal timing of repeat cell-free DNA testing in cases with an initial screen failure. STUDY DESIGN: This was a retrospective cohort study of women undergoing cell-free DNA for fetal aneuploidy screening between 9 and 18 weeks through a single laboratory over 1 year from 2018 to 2019. Fetal fraction change per week was calculated, and generalized linear models were used to calculate relative risk and 95% confidence interval of a no call result at given maternal weights and gestational ages. RESULTS: The vast majority of samples (99.22%) received a test result. The risk of a no call result owing to a low fetal fraction was higher with increasing maternal weight. At 9 to 12 weeks, the rate of a no call result owing to a low fetal fraction in women who weighed <150 lb was 0.14% compared with 17.39% in women weighing >400 lb. Fetal fraction increased with increasing gestational age, although the incremental increase in fetal fraction over time is inversely proportional to maternal weight. At 13 to 18 weeks' gestation, 6.45% of women weighing >400 lb received a no call result owing to a low fetal fraction. In women in the highest weight category, >400 lb, fetal fraction increased 0.5% with each week of gestation. CONCLUSION: Although the risk of a no call result increases with maternal weight, cell-free DNA screening should be offered to all women at 9 to 12 weeks' gestation, allowing the option to have chorionic villus sampling after a positive test result. Pretest counseling for women with obesity should include the increased chance for a test failure. Most women weighing less than 400 lb will receive a test result and more than 80% of women with a weight of >400 lb will receive a test result at 9 to 12 weeks' gestation. Data regarding the expected increase in cell-free DNA fetal fraction per week may help guide the timing of a redraw to optimize test success.

Not all low fetal fraction cell-free DNA screening failures are at increased risk for aneuploidy.

OBJECTIVE: To evaluate cell-free DNA (cfDNA) redraws and pregnancy outcomes following low fetal fraction (FF) cfDNA failures, as it has been suggested that a failed cfDNA screen due to insufficient FF carries increased risk for fetal aneuploidy. METHODS: Here >200,000 consecutive samples were reviewed and >1,100 patients were identified with a failed cfDNA due to low FF using genome-wide massively parallel sequencing. Redraw results following the initial low FF failure were analyzed, as well as pregnancy outcomes for patients with repeated low FF failure on redraw. RESULTS: Upon redraw 84.2% of samples yielded a reportable result with no enrichment of aneuploidy observed (p = 0.332). Higher maternal weights and multifetal pregnancy rates were observed in samples with insufficient FF. In patients with repeated low FF failure on redraw, almost all pregnancies resulted in apparently healthy liveborns. CONCLUSION: Insufficient FF was not an indicator of aneuploidy risk or adverse pregnancy outcomes in this study. Caution should be taken in generalizing aneuploidy risk to all low FF cfDNA failures. Redrawing may be an appropriate next step, as proceeding directly with diagnostic testing for aneuploidy may be unwarranted for most patients.

Impact of mosaicism ratio on positive predictive value of cfDNA screening.

OBJECTIVE: To examine the relationship between the fraction of cell-free DNA (cfDNA) affected by aneuploidy compared to the overall fetal fraction of a prenatal screening specimen and its effect on positive predictive value (PPV). METHOD: CfDNA specimens positive for trisomy 13, 18, and 21 with diagnostic outcomes were analysed over a 22-month period in one clinical laboratory. For each positive specimen, a "mosaicism ratio" (MR) was calculated by dividing the fraction of cfDNA affected by aneuploidy by the overall fetal fraction of the specimen. PPVs were calculated and analyzed based on various MR ranges. RESULTS: Trisomy 13 was the aneuploidy most commonly seen in mosaic form, followed by trisomy 18 and trisomy 21. Significant differences in positive predictive values were noted for all three trisomies between samples with an MR in the "mosaic" versus "non-mosaic" range, as well as between results classified as "low-mosaic" versus "high-mosaic." CONCLUSION: PPVs may be influenced, in part, by the mosaicism ratio associated with a particular result. The data generated from this study may be useful in providing more personalized risk assessments for patients with positive cfDNA screening results.

Confirmatory testing illustrates additional risks for structural sex chromosome abnormalities in fetuses with a non-invasive prenatal screen positive for monosomy X.

More and more women rely on non-invasive prenatal screening (NIPS) to detect fetal sex and risk for aneuploidy. The testing applies massively parallel sequencing or single nucleotide polymorphism (SNP) microarray to circulating cell-free DNA to determine relative copy number. In addition to trisomies 13, 18, and 21, some labs offer screening for sex chromosome abnormalities as part of their test. In this study, an index neonate screened positive for monosomy X and had discordant postnatal chromosomes indicating an X;autosome translocation. This patient prompted a retrospective chart review for similar cases at a large NIPS testing center. The review found 28 patients with an abnormal NIPS for monosomy X who were eventually diagnosed with additional discrepant structural sex chromosome abnormalities including translocations, isochromosomes, deletions, rings, markers, and uniparental disomy. The majority of these were mosaic with monosomy X, but in seven cases, there was no evidence of mosaicism on confirmatory testing. The identification of multiple sex chromosome aneuploidies in these cases supports the need for additional genetic counseling prior to NIPS testing and following abnormal NIPS results that are positive for monosomy X. This finding broadens our knowledge about the variable outcomes of positive monosomy X NIPS results and emphasizes the importance of confirmatory testing and clinical follow up for these patients.

Deletion rescue resulting in segmental homozygosity: A mechanism underlying discordant NIPT results.

With the increasing capabilities of non-invasive prenatal testing (NIPT), detection of sub-chromosomal deletions and duplications are possible. This case series of deletion rescues resulting in segmental homozygosity helps provide a biological explanation for NIPT discrepancies and adds to the dearth of existing literature surrounding segmental UPD cases and their underlying mechanisms. In the three cases presented here, NIPT reported a sub-chromosomal deletion (in isolation or as part of a complex finding). Diagnostic testing, however, revealed segmental homozygosity or UPD for the region reported deleted on NIPT. Postnatal placental testing was pursued in two cases and confirmed the NIPT findings. This discordance between the screening and diagnostic testing is suggestive of a corrective post-zygotic event, such as telomere capture and/or deletion rescue, ultimately resulting in segmental homozygosity and fetoplacental mosaicism. Imprinted chromosomes and autosomal recessive disease genes make homozygosity an important clinical consideration. Amniocentesis with SNP microarray is particularly useful in determining both copy number and UPD issues alike.

A Systematic Review of Randomized Controlled Trials to Assess Outcomes of Genetic Counseling.

With the advancements in precision medicine and health care reform, it is critical that genetic counseling practice respond to emerging evidence to maximize client benefit. The objective of this review was to synthesize evidence on outcomes from randomized controlled trials (RCTs) of genetic counseling to inform clinical practice. Seven databases were searched in conducting this review. Studies were selected for inclusion if they were: (a) RCTs published from 1990 to 2015, and (b) assessed a direct outcome of genetic counseling. Extracted data included study population, aims, and outcomes. Risk of bias was evaluated using the Cochrane Handbook for Systematic Reviews of Interventions guidelines. A review of 1654 abstracts identified 58 publications of 54 unique RCTs that met inclusion criteria, the vast majority of which were conducted in cancer genetic counseling setting. Twenty-seven publications assessed 'enhancements' to genetic counseling, and 31 publications compared delivery modes. The methodological rigor varied considerably, highlighting the need for attention to quality criteria in RCT design. While most studies assessed several client outcomes hypothesized to be affected by genetic counseling (e.g., psychological wellbeing, knowledge, perceived risk, patient satisfaction), disparate validated and reliable scales and other assessments were often used to evaluate the same outcome(s). This limits opportunity to compare findings across studies. While RCTs of genetic counseling demonstrate enhanced client outcomes in a number of studies and pave the way to evidence-based practice, the heterogeneity of the research questions suggest an important need for more complementary studies with consistent outcome assessments.

Participant use and communication of findings from exome sequencing: a mixed-methods study.

PURPOSE: This study investigated how genome sequencing results affect health behaviors, affect, and communication. METHODS: We report on 29 participants who received a sequence result in the ClinSeq study, a cohort of well-educated, postreproductive volunteers. A mixed-methods design was used to explore respondents' use, communication, and perceived utility of results. RESULTS: Most participants (72%) shared their result with at least one health-care provider, and 31% reported subsequent changes in the health care they received. Participants scored high on the Positive Experiences subscale and low on the Distress subscale of a modified version of the Multidimensional Impact of Cancer Risk Assessment. The majority (93%) shared their result with at least one family member. Participants described deriving personal utility from their results. CONCLUSION: This article is the first to describe research participants' reactions to actionable sequencing results. Our findings suggest clinical and personal benefit from receiving sequencing results, both of which may contribute to improved health for the recipients. Given the participants' largely positive or neutral affective responses and disclosure of their results to physicians and relatives, health-care providers should redirect concern from the potential for distress and attend to motivating patients to follow their medical recommendations.Genet Med 18 6, 577-583.

Prenatal cfDNA Screening for Emanuel Syndrome and Other Unbalanced Products of Conception in Carriers of the Recurrent Balanced Translocation t(11;22): One Laboratory's Retrospective Experience.

Prenatal cell-free DNA screening (cfDNA) can identify fetal chromosome abnormalities beyond common trisomies. Emanuel syndrome (ES), caused by an unbalanced translocation between chromosomes 11 and 22, has lacked a reliable prenatal screening option for families with a carrier parent. A cohort of cases (n = 46) sent for cfDNA screening with indications and/or results related to ES was queried; diagnostic testing and pregnancy outcomes were requested and analyzed. No discordant results were reported or suspected; there were ten true positives with diagnostic confirmation, six likely concordant positives based on known translocations and consistent cfDNA data, and twenty-six true negatives, by diagnostic testing or birth outcomes. For cases with parental testing, all affected ES cases had maternal translocation carriers. Expanded cfDNA may provide reassurance for t(11;22) carriers with screen negative results, and screen positive results appear to reflect a likely affected fetus, especially with a known maternal translocation. Current society guidelines support the use of expanded cfDNA screening in specific circumstances, such as for translocation carriers, with appropriate counseling. Diagnostic testing is recommended for prenatal diagnosis of ES and other chromosome abnormalities in pregnancy. To our knowledge, this cohort is the largest published group of cases with prenatal screening for carriers of t(11;22).

Comprehensive Metabolic Tracing Reveals the Origin and Catabolism of Cysteine in Mammalian Tissues and Tumors.

Cysteine plays critical roles in cellular biosynthesis, enzyme catalysis, and redox metabolism. The intracellular cysteine pool can be sustained by cystine uptake or de novo synthesis from serine and homocysteine. Demand for cysteine is increased during tumorigenesis for generating glutathione to deal with oxidative stress. While cultured cells have been shown to be highly dependent on exogenous cystine for proliferation and survival, how diverse tissues obtain and use cysteine in vivo has not been characterized. We comprehensively interrogated cysteine metabolism in normal murine tissues and cancers that arise from them using stable isotope 13C1-serine and 13C6-cystine tracing. De novo cysteine synthesis was highest in normal liver and pancreas and absent in lung tissue, while cysteine synthesis was either inactive or downregulated during tumorigenesis. In contrast, cystine uptake and metabolism to downstream metabolites was a universal feature of normal tissues and tumors. However, differences in glutathione labeling from cysteine were evident across tumor types. Thus, cystine is a major contributor to the cysteine pool in tumors, and glutathione metabolism is differentially active across tumor types. SIGNIFICANCE: Stable isotope 13C1-serine and 13C6-cystine tracing characterizes cysteine metabolism in normal murine tissues and its rewiring in tumors using genetically engineered mouse models of liver, pancreas, and lung cancers.

Distinct Nrf2 Signaling Thresholds Mediate Lung Tumor Initiation and Progression.

Mutations in the KEAP1-NRF2 (Kelch-like ECH-associated protein 1-nuclear factor-erythroid 2 p45-related factor 2) pathway occur in up to a third of non-small cell lung cancer (NSCLC) cases and often confer resistance to therapy and poor outcomes. Here, we developed murine alleles of the KEAP1 and NRF2 mutations found in human NSCLC and comprehensively interrogated their impact on tumor initiation and progression. Chronic NRF2 stabilization by Keap1 or Nrf2 mutation was not sufficient to induce tumorigenesis, even in the absence of tumor suppressors, p53 or LKB1. When combined with KrasG12D/+, constitutive NRF2 activation promoted lung tumor initiation and early progression of hyperplasia to low-grade tumors but impaired their progression to advanced-grade tumors, which was reversed by NRF2 deletion. Finally, NRF2 overexpression in KEAP1 mutant human NSCLC cell lines was detrimental to cell proliferation, viability, and anchorage-independent colony formation. Collectively, these results establish the context-dependence and activity threshold for NRF2 during the lung tumorigenic process. SIGNIFICANCE: Stabilization of the transcription factor NRF2 promotes oncogene-driven tumor initiation but blocks tumor progression, indicating distinct, threshold-dependent effects of the KEAP1/NRF2 pathway in different stages of lung tumorigenesis.

Clinical significance and mechanisms associated with segmental UPD.

Whole chromosome uniparental disomy (UPD) has been well documented with mechanisms largely understood. However, the etiology of segmental limited UPD (segUPD) is not as clear. In a 10-year period of confirming (> 300) cases of whole chromosome UPD, we identified 86 segmental cases in both prenatal and postnatal samples. Thirty-two of these cases showed mosaic segmental UPD at 11p due to somatic selection associated with Beckwith-Wiedemann syndrome. This study focuses on apparent mechanisms associated with the remaining cases, many of which appear to represent corrections of genomic imbalance such as deletions and derivative chromosomes. In some cases, segmental UPD was associated with the generation of additional genomic imbalance while in others it apparently resulted in restoration of euploidy. Multiple tests utilizing noninvasive prenatal testing (NIPT), chorionic villus sampling (CVS) and amniotic fluid samples from the same pregnancy revealed temporal evidence of correction and a "hotspot" at 1p. Although in many cases the genomic imbalance was dosage "repaired" in the analyzed tissue, clinical effects could be sustained due to early developmental effects of the original imbalance or due to its continued existence in other tissues. In addition, if correction did not occur in the gametes there would be recurrence risks for the offspring of those individuals. Familial microarray allele patterns are presented that differentiate lack of gamete correction from somatic derived gonadal mosaicism. These results suggest that the incidence of segUPD mediated correction is underestimated and may explain the etiology of some clinical phenotypes which are undetected by routine microarray analysis and many exome sequencing studies.

Application of mosaicism ratio to multifetal gestations.

Mosaicism ratio, or MR, is a laboratory metric that can be calculated using massively parallel sequencing data from cell-free DNA (cfDNA) screening. MR compares the amount of cfDNA present from a particular chromosome or chromosomal region to the overall fetal fraction of the specimen. In singleton gestations, MR may be used to refine the positive predictive value of an abnormal cfDNA screening result by identifying cases that could be impacted by various biological factors, such as placental mosaicism or prior co-twin demise. The current study was designed to examine the behavior of mosaicism ratio (MR) in multifetal gestations. Multifetal cfDNA specimens with positive results for trisomies 21, 18, or 13 and confirmed diagnostic outcomes were compiled to examine MR of the aneuploid chromosome based on the number of affected fetuses/placentas. A second multifetal cohort was assembled to analyze the MR of the Y chromosome in cases with at least one male fetus. For aneuploid cases, the average MR of affected singletons (used as a biological proxy for two affected twins) was significantly higher than the average MR for twins in which one fetus was affected. The average MR of the aneuploid chromosome for one affected twin was 52%, 42%, and 48% of that of singleton gestations for trisomy 21, 18, and 13 cases, respectively. MR cutoffs of 0.7 for trisomy 21, and 0.5 for trisomies 18 and 13 may help predict whether one versus both twins are affected with aneuploidy when clinical concern arises. For male cases, the Y MR of XX/XY gestations was 48% of the Y MR for XY/XY gestations. Using a Y MR cutoff of 0.8 allowed determination of XX/XY versus XY/XY gestations with 92.3-94.9% accuracy. Based on the data presented, MR may have utility in the analysis and interpretation of cfDNA data from multifetal gestations.

Factors influencing time to determination of the recommended phase 2 dose in phase 1 clinical trials.

OBJECTIVE: Cancer clinical trials are the means to develop safe and effective novel treatment options for patients. The longer it takes for these trials to reach the recommended phase 2 dose (RP2D), the fewer therapy options are available to patients and physicians. The purpose of this study is to examine the factors that delay RP2D determination in phase 1 clinical trials. METHODS: Thirty-five consecutive phase 1 clinical trials for advanced solid tumors that started between February 2006 and March 2009 in a single institution were examined for inclusion. Factors potentially contributing to trial delays were analyzed against time to determination of an RP2D (TDR). RESULTS: Thirty-one phase 1 clinical trials met the inclusion criteria and were included in the statistical analysis. Investigational agents under evaluation included single agent cytotoxic (N=4), monoclonal antibody (N=3), single agent cytostatic/targeted (N=16), or combination of an investigational agent and commercially available systemic chemotherapy (N=8). A protocol defined phase 2 dose decreased the TDR (P<0.001). Other factors that significantly increased TDR included a larger minimum estimated patient sample size (P=0.022), a greater number of predefined dose levels (P<0.001), and a higher number of expansion cohorts (P=0.038). CONCLUSIONS: Including a predefined phase 2 dose and reducing the number of dose levels and expansion cohorts may shorten phase 1 trial TDR.

Executive functioning and gambling: performance on the trail making test is associated with gambling problems in older adult gamblers.

Rates of gambling problems in older adults have risen with increased accessibility of gambling venues. One possible contributor to problem gambling among older adults is decreased self-control brought about by diminished executive functioning. Consistent with this possibility, Study 1 revealed that older adults recruited from gambling venues reported greater gambling problems if they also experienced deficits in executive functioning, measured via the Trail Making Test. Study 2 replicated this finding and demonstrated that problem gambling is associated with increased depression among older adults, mediated by increased financial distress. These studies provide support for the hypothesis that older adult gamblers who have executive functioning problems are also likely to have gambling problems.