Accuracy of fetal fraction measurements in a single-nucleotide polymorphism-based noninvasive prenatal test.

BACKGROUND: Noninvasive prenatal testing (NIPT) for fetal aneuploidy relies on the analysis of fetoplacental cell-free DNA (cfDNA) found in maternal plasma. A minimum cfDNA fetal fraction (FF) is required for reliable test performance, but some methods may have suboptimal accuracy for FF measurement. This study investigated the accuracy of a single-nucleotide polymorphism- (SNP-) based NIPT method to assess FF. METHODS: FF measurements using SNP-based NIPT in consecutive samples from singleton male pregnancies were compared with FF measured using a "gold standard" Y-chromosome method. RESULTS: In a cohort of 106,846 samples, the SNP-based FF method showed a standard deviation (SD) of 0.42%. Compared to the Y chromosome FF method, a correlation coefficient, r, of 0.995, and bias of 0.17% were observed. The SD was not substantially different across specific FF ranges or for samples with high-risk NIPT results. CONCLUSIONS: The SNP-based NIPT method estimates FF with good accuracy, with a SD three to eight times better than other NIPT methods (0.42% vs. 1.3%-3.4%). FF is an important quality control parameter and should be routinely reported as part of NIPT.

Elevation of Donor-derived Cell-free DNA Before Biopsy-proven Rejection in Kidney Transplant.

BACKGROUND: Standard-of-care biomarkers for renal allograft rejection are lagging indicators, signaling existing organ injury. This precludes early intervention, when immunological cascades leading to rejection are most susceptible. Donor-derived cell-free DNA (dd-cfDNA) shows promise as an early indicator of rejection, allowing earlier and possibly more effective treatment. This analysis was designed to assess this promise using real-world dd-cfDNA testing evidence. METHODS: This retrospective analysis of the prospective, observational ProActive registry study (NCT04091984) assessed dd-cfDNA and serum creatinine levels before biopsy in 424 patients with ≥1 dd-cfDNA test (n = 1013) in the 6 mo before biopsy. RESULTS: Of 4667 enrolled patients, 1631 patients had ≥18 mo of follow-up data, of which 424 had a biopsy and were included in this analysis. Twenty-six biopsies showed antibody-mediated rejection (ABMR), 62 showed T cell-mediated rejection, and 336 showed nonrejection; each from a unique patient. dd-cfDNA fractions were significantly elevated 5 mo before ABMR biopsies, and 2 mo before T cell-mediated rejection biopsies, compared with nonrejection biopsies. In contrast, serum creatinine did not discriminate between rejection and nonrejection in advance, or concurrent with biopsy. Among patients with nonrejection biopsies, estimated glomerular filtration rate was significantly lower in cases with ≥2 increased dd-cfDNA results (≥1%), compared with those with 0 or 1 increased dd-cfDNA result. CONCLUSIONS: These data indicate that dd-cfDNA is an early indicator of biopsy-proven rejection, especially ABMR, suggesting a greater role for dd-cfDNA in surveillance to identify patients at high risk of ongoing or future rejection, thus requiring closer monitoring, biopsy, or other management changes.

Use of Donor-derived Cell-free DNA to Inform Tapering of Immunosuppression Therapy in Kidney Transplant Recipients: An Observational Study.

Background: Immunosuppression therapy (IST) is required for allograft survival but can cause significant adverse effects. Donor-derived cell-free DNA (dd-cfDNA) is a validated noninvasive biomarker for active rejection in kidney transplant (KTx). Evidence supporting dd-cfDNA testing use in IST management is limited. Methods: In this single-center observational study, dd-cfDNA testing was performed in 21 KTx patients considered good candidates for mycophenolic acid (MPA) reduction. Patients with dd-cfDNA <1% at the first visit (enrollment) had their MPA dosage reduced; those with dd-cfDNA ≥1% had their MPA dosage maintained. Patients were monitored with dd-cfDNA for 6 additional visits. Results: Of 21 patients enrolled in the study, 17 were considered low risk for rejection by dd-cfDNA and underwent MPA reduction; 4 patients were considered high risk for rejection by dd-cfDNA and had their initial MPA dosage maintained. Of the 4 patients considered high risk for rejection by dd-cfDNA, 1 experienced chronic allograft nephropathy and graft loss, and another received an indication biopsy that showed no evidence of rejection. Of the 17 patients considered low risk for rejection by dd-cfDNA, none experienced allograft rejection. dd-cfDNA was used for surveillance in a 6-mo period following MPA reduction; no untoward results were noted. Conclusions: This proof-of-concept study reports the use of dd-cfDNA to directly inform IST management in a cohort of KTx who were candidates for IST reduction.

Comparing Plasma Donor-derived Cell-free DNA to Gene Expression in Endomyocardial Biopsies in the Trifecta-Heart Study.

BACKGROUND: Plasma donor-derived cell-free DNA (dd-cfDNA) is used to screen for rejection in heart transplants. We launched the Trifecta-Heart study (ClinicalTrials.gov No. NCT04707872), an investigator-initiated, prospective trial, to examine the correlations between genome-wide molecular changes in endomyocardial biopsies (EMBs) and plasma dd-cfDNA. The present report analyzes the correlation of plasma dd-cfDNA with gene expression in EMBs from 4 vanguard centers and compared these correlations with those in 604 kidney transplant biopsies in the Trifecta-Kidney study (ClinicalTrials.gov No. NCT04239703). METHODS: We analyzed 137 consecutive dd-cfDNA-EMB pairs from 70 patients. Plasma %dd-cfDNA was measured by the Prospera test (Natera Inc), and gene expression in EMBs was assessed by Molecular Microscope Diagnostic System using machine-learning algorithms to interpret rejection and injury states. RESULTS: Top transcripts correlating with dd-cfDNA were related to genes increased in rejection such as interferon gamma-inducible genes (eg, HLA-DMA ) but also with genes induced by injury and expressed in macrophages (eg, SERPINA1 and HMOX1 ). In gene enrichment analysis, the top dd-cfDNA-correlated genes reflected inflammation and rejection pathways. Dd-cfDNA correlations with rejection genes in EMB were similar to those seen in kidney transplant biopsies, with somewhat stronger correlations for TCMR genes in hearts and ABMR genes in kidneys. However, the correlations with parenchymal injury-induced genes and macrophage genes were much stronger in hearts. CONCLUSIONS: In this first analysis of Trifecta-Heart study, dd-cfDNA correlates significantly with molecular rejection but also with injury and macrophage infiltration, reflecting the proinflammatory properties of injured cardiomyocytes. The relationship supports the utility of dd-cfDNA in clinical management of heart transplant recipients.

Distinct Molecular Processes Mediate Donor-derived Cell-free DNA Release From Kidney Transplants in Different Disease States.

BACKGROUND: Among all biopsies in the Trifecta-Kidney Study ( ClinicalTrials.gov NCT04239703), elevated plasma donor-derived cell-free DNA (dd-cfDNA) correlated most strongly with molecular antibody-mediated rejection (AMR) but was also elevated in other states: T cell-mediated rejection (TCMR), acute kidney injury (AKI), and some apparently normal biopsies. The present study aimed to define the molecular correlates of plasma dd-cfDNA within specific states. METHODS: Dd-cfDNA was measured by the Prospera test. Molecular rejection and injury states were defined using the Molecular Microscope system. We studied the correlation between dd-cfDNA and the expression of genes, transcript sets, and classifier scores within specific disease states, and compared AMR, TCMR, and AKI to biopsies classified as normal and no injury (NRNI). RESULTS: In all 604 biopsies, dd-cfDNA was elevated in AMR, TCMR, and AKI. Within AMR biopsies, dd-cfDNA correlated with AMR activity and stage. Within AKI, the correlations reflected acute parenchymal injury, including cell cycling. Within biopsies classified as MMDx Normal and archetypal No injury (NRNI), dd-cfDNA still correlated significantly with rejection- and injury-related genes. TCMR activity (eg, the TCMR Prob classifier) correlated with dd-cfDNA, but within TCMR biopsies, top gene correlations were complex and not the top TCMR-selective genes. CONCLUSIONS: In kidney transplants, elevated plasma dd-cfDNA is associated with 3 distinct molecular states in the donor tissue: AMR, recent parenchymal injury (including cell cycling), and TCMR, potentially complicated by parenchymal disruption. Moreover, subtle rejection- and injury-related changes in the donor tissue can contribute to dd-cfDNA elevations in transplants considered to have no rejection or injury.

Impact of high-risk prenatal screening results for 22q11.2 deletion syndrome on obstetric and neonatal management: Secondary analysis from the SMART study.

OBJECTIVE: One goal of prenatal genetic screening is to optimize perinatal care and improve infant outcomes. We sought to determine whether high-risk cfDNA screening for 22q11.2 deletion syndrome (22q11.2DS) affected prenatal or neonatal management. METHODS: This was a secondary analysis from the SMART study. Patients with high-risk cfDNA results for 22q11.2DS were compared with the low-risk cohort for pregnancy characteristics and obstetrical management. To assess differences in neonatal care, we compared high-risk neonates without prenatal genetic confirmation with a 1:1 matched low-risk cohort. RESULTS: Of 18,020 eligible participants enrolled between 2015 and 2019, 38 (0.21%) were high-risk and 17,982 (99.79%) were low-risk for 22q11.2DS by cfDNA screening. High-risk participants had more prenatal diagnostic testing (55.3%; 21/38 vs. 2.0%; 352/17,982, p < 0.001) and fetal echocardiography (76.9%; 10/13 vs. 19.6%; 10/51, p < 0.001). High-risk newborns without prenatal diagnostic testing had higher rates of neonatal genetic testing (46.2%; 6/13 vs. 0%; 0/51, P < 0.001), echocardiography (30.8%; 4/13 vs. 4.0%; 2/50, p = 0.013), evaluation of calcium levels (46.2%; 6/13 vs. 4.1%; 2/49, P < 0.001) and lymphocyte count (53.8%; 7/13 vs. 15.7%; 8/51, p = 0.008). CONCLUSIONS: High-risk screening results for 22q11.2DS were associated with higher rates of prenatal and neonatal diagnostic genetic testing and other 22q11.2DS-specific evaluations. However, these interventions were not universally performed, and >50% of high-risk infants were discharged without genetic testing, representing possible missed opportunities to improve outcomes for affected individuals.

The Clinical Utility of Genetic Testing in the Diagnosis and Management of Adults with Chronic Kidney Disease.

SIGNIFICANCE STATEMENT: Accurate diagnosis of a patient's underlying cause of CKD can influence management and ultimately overall health. The single-arm, interventional, prospective Renasight Clinical Application, Review, and Evaluation study assessed the utility of genetic testing with a 385 gene kidney disease panel on the diagnosis and management of 1623 patients with CKD. Among 20.8% of patients who had positive genetic findings, half resulted in a new or reclassified diagnosis. In addition, a change in management because of genetic testing was reported for 90.7% of patients with positive findings, including treatment changes in 32.9%. These findings demonstrate that genetic testing has a significant effect on both CKD diagnosis and management. BACKGROUND: Genetic testing in CKD has recently been shown to have diagnostic utility with many predicted implications for clinical management, but its effect on management has not been prospectively evaluated. METHODS: Renasight Clinical Application, Review, and Evaluation RenaCARE (ClinicalTrials.gov NCT05846113 ) is a single-arm, interventional, prospective, multicenter study that evaluated the utility of genetic testing with a broad, 385 gene panel (the Renasight TM test) on the diagnosis and management of adult patients with CKD recruited from 31 US-based community and academic medical centers. Patient medical history and clinical CKD diagnosis were collected at enrollment. Physician responses to questionnaires regarding patient disease categorization and management were collected before genetic testing and 1 month after the return of test results. Changes in CKD diagnosis and management after genetic testing were assessed. RESULTS: Of 1623 patients with CKD in 13 predefined clinical disease categories (ages, 18-96; median, 55 years), 20.8% ( n =338) had positive genetic findings spanning 54 genes. Positive genetic findings provided a new diagnosis or reclassified a prior diagnosis in 48.8% of those patients. Physicians reported that genetic results altered the management of 90.7% of patients with a positive genetic finding, including changes in treatment plan, which were reported in 32.9% of these patients. CONCLUSIONS: Genetic testing with a CKD-focused 385 gene panel substantially refined clinical diagnoses and had widespread implications for clinical management, including appropriate treatment strategies. These data support the utility of broader integration of panels of genetic tests into the clinical care paradigm for patients with CKD. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: ClinicalTrials.gov, NCT05846113 .

Performance of prenatal cfDNA screening for sex chromosomes.

PURPOSE: The aim of this study was to assess the performance of cell-free DNA (cfDNA) screening to detect sex chromosome aneuploidies (SCAs) in an unselected obstetrical population with genetic confirmation. METHODS: This was a planned secondary analysis of the multicenter, prospective SNP-based Microdeletion and Aneuploidy RegisTry (SMART) study. Patients receiving cfDNA results for autosomal aneuploidies and who had confirmatory genetic results for the relevant sex chromosomal aneuploidies were included. Screening performance for SCAs, including monosomy X (MX) and the sex chromosome trisomies (SCT: 47,XXX; 47,XXY; 47,XYY) was determined. Fetal sex concordance between cfDNA and genetic screening was also evaluated in euploid pregnancies. RESULTS: A total of 17,538 cases met inclusion criteria. Performance of cfDNA for MX, SCTs, and fetal sex was determined in 17,297, 10,333, and 14,486 pregnancies, respectively. Sensitivity, specificity, and positive predictive value (PPV) of cfDNA were 83.3%, 99.9%, and 22.7% for MX and 70.4%, 99.9%, and 82.6%, respectively, for the combined SCTs. The accuracy of fetal sex prediction by cfDNA was 100%. CONCLUSION: Screening performance of cfDNA for SCAs is comparable to that reported in other studies. The PPV for the SCTs was similar to the autosomal trisomies, whereas the PPV for MX was substantially lower. No discordance in fetal sex was observed between cfDNA and postnatal genetic screening in euploid pregnancies. These data will assist interpretation and counseling for cfDNA results for sex chromosomes.

Obstetrical, perinatal, and genetic outcomes associated with nonreportable prenatal cell-free DNA screening results.

BACKGROUND: The clinical implications of nonreportable cell-free DNA screening results are uncertain, but such results may indicate poor placental implantation in some cases and be associated with adverse obstetrical and perinatal outcomes. OBJECTIVE: This study aimed to assess the outcomes of pregnancies with nonreportable cell-free DNA screening in a cohort of patients with complete genetic and obstetrical outcomes. STUDY DESIGN: This was a prespecified secondary analysis of a multicenter prospective observational study of prenatal cell-free DNA screening for fetal aneuploidy and 22q11.2 deletion syndrome. Participants who underwent cell-free DNA screening from April 2015 through January 2019 were offered participation. Obstetrical outcomes and neonatal genetic testing results were collected from 21 primary-care and referral centers in the United States, Europe, and Australia. The primary outcome was risk for adverse obstetrical and perinatal outcomes (aneuploidy, preterm birth at <28, <34, and <37 weeks' gestation, preeclampsia, small for gestational age or birthweight <10th percentile for gestational week, and a composite outcome that included preterm birth at <37 weeks, preeclampsia, small for gestational age, and stillbirth at >20 weeks) after nonreportable cell-free DNA screening because of low fetal fraction or other causes. Multivariable analyses were performed, adjusting for variables known to be associated with obstetrical and perinatal outcomes, nonreportable results, or fetal fraction. RESULTS: In total, 25,199 pregnant individuals were screened, and 20,194 were enrolled. Genetic confirmation was missing in 1165 (5.8%), 1085 (5.4%) were lost to follow-up, and 93 (0.5%) withdrew; the final study cohort included 17,851 (88.4%) participants who had cell-free DNA, fetal or newborn genetic confirmatory testing, and obstetrical and perinatal outcomes collected. Results were nonreportable in 602 (3.4%) participants. A sample was redrawn and testing attempted again in 427; in 112 (26.2%) participants, results were again nonreportable. Nonreportable results were associated with higher body mass index, chronic hypertension, later gestational age, lower fetal fraction, and Black race. Trisomy 13, 18, or 21 was confirmed in 1.6% with nonreportable tests vs 0.7% with reported results (P=.013). Rates of preterm birth at <28, 34, and 37 weeks, preeclampsia, and the composite outcome were higher among participants with nonreportable results, and further increased among those with a second nonreportable test, whereas the rate of small for gestational age infants was not increased. After adjustment for confounders, the adjusted odds ratios were 2.2 (95% confidence interval, 1.1-4.4) and 2.6 (95% confidence interval, 0.6-10.8) for aneuploidy, and 1.5 (95% confidence interval, 1.2-1.8) and 2.1 (95% confidence interval, 1.4-3.2) for the composite outcome after a first and second nonreportable test, respectively. Of the patients with nonreportable tests, 94.9% had a live birth, as opposed to 98.8% of those with reported test results (adjusted odds ratio for livebirth, 0.20 [95% confidence interval, 0.13-0.30]). CONCLUSION: Patients with nonreportable cell-free DNA results are at increased risk for a number of adverse outcomes, including aneuploidy, preeclampsia, and preterm birth. They should be offered diagnostic genetic testing, and clinicians should be aware of the increased risk of pregnancy complications.

Antibody-mediated Rejection Without Detectable Donor-specific Antibody Releases Donor-derived Cell-free DNA: Results From the Trifecta Study.

BACKGROUND: Trifecta (ClinicalTrials.gov #NCT04239703) is a prospective trial defining relationships between donor-derived cell-free DNA (dd-cfDNA), donor-specific antibody (DSA), and molecular findings in kidney transplant biopsies. Previous analyses of double results showed dd-cfDNA was strongly associated with rejection-associated molecules in the biopsy. The present study analyzed the triple results in 280 biopsies, focusing on the question of dd-cfDNA levels in DSA-negative antibody-mediated rejection (AMR). METHODS: Molecular Microscope Diagnostic System biopsy testing was performed at Alberta Transplant Applied Genomics Centre, dd-cfDNA testing at Natera, Inc, and central HLA antibody testing at One Lambda Inc. Local DSA and histologic diagnoses were assigned per center standard-of-care. RESULTS: DSA was frequently negative in both molecular (56%) and histologic (51%) AMR. DSA-negative AMR had slightly less molecular AMR activity and histologic peritubular capillaritis than DSA-positive AMR. However, all AMRs-DSA-positive or -negative-showed elevated %dd-cfDNA. There was no association between dd-cfDNA and DSA in biopsies without rejection. In AMR, %dd-cfDNA ≥1.0 was more frequent (75%) than DSA positivity (44%). In logistic regression, dd-cfDNA percent (area under the curve [AUC] 0.85) or quantity (AUC 0.86) predicted molecular AMR better than DSA (AUC 0.66). However, the best predictions incorporated both dd-cfDNA and DSA, plus time posttransplant (AUC 0.88). CONCLUSIONS: DSA-negative AMR has moderately decreased mean molecular and histologic AMR-associated features compared with DSA-positive AMR, though similarly elevated dd-cfDNA levels. In predicting AMR at the time of indication biopsies in this population, dd-cfDNA is superior to DSA, reflecting the prevalence of DSA-negative AMR, but the optimal predictions incorporated both dd-cfDNA and DSA.

Development of a Novel Method of Assessing Potential Loss of Control in Internet Gaming Disorder.

As video game play has increased in popularity, so too have reports that a subset of individuals play games in a way that causes negative consequences to their lives, which has resulted in the proposed inclusion of Internet Gaming Disorder (IGD) as a formal mental health diagnosis. Given the mass popularity of video games, it is critical that screening materials for this proposed disorder are sufficiently sensitive to ensure that individuals who suffer harm are identified, while those who do not are not mislabeled as such. Here we examined the extent to which participants' responses to a typical IGD questionnaire predicted academic behaviors that could be associated with harm. We recruited 42 college students and tracked their gaming and studying habits weekly over the course of one semester, taking particular note of weeks in which participants did or did not have exams. We predicted that college students overall would spend less time engaging with video games and more time with academics when they had exams, but such modulations would be smaller among individuals with more initial IGD symptoms (i.e., the "more IGD symptoms" group, as compared to the "fewer IGD symptoms" group, would show loss of control). We did not find that college students overall spent less time engaging with video games and more time with academics when they had exams, but post-hoc effect size analyses indicated that our study was underpowered. Implications of potential results using this methodology are discussed and estimates of powerful sample sizes are provided.

Assessment of an automated approach for variant interpretation in screening for monogenic disorders: A single-center study.

BACKGROUND: Automation has been introduced into variant interpretation, but it is not known how automated variant interpretation performs on a stand-alone basis. The purpose of this study was to evaluate a fully automated computerized approach. METHOD: We reviewed all variants encountered in a set of carrier screening panels over a 1-year interval. Observed variants with high-confidence ClinVar interpretations were included in the analysis; those without high-confidence ClinVar entries were excluded. RESULTS: Discrepancy rates between automated interpretations and high-confidence ClinVar entries were analyzed. Of the variants interpreted as positive (likely pathogenic or pathogenic) based on ClinVar information, 22.6% were classified as negative (variants of uncertain significance, likely benign or benign) variants by the automated method. Of the ClinVar negative variants, 1.7% were classified as positive by the automated software. On a per-case basis, which accounts for variant frequency, 63.4% of cases with a ClinVar high-confidence positive variant were classified as negative by the automated method. CONCLUSION: While automation in genetic variant interpretation holds promise, there is still a need for manual review of the output. Additional validation of automated variant interpretation methods should be conducted.

A Critical Evaluation of Validation and Clinical Experience Studies in Non-Invasive Prenatal Testing for Trisomies 21, 18, and 13 and Monosomy X.

Non-invasive prenatal testing (NIPT) for trisomies 21, 18, 13 and monosomy X is widely utilized with massively parallel shotgun sequencing (MPSS), digital analysis of selected regions (DANSR), and single nucleotide polymorphism (SNP) analyses being the most widely reported methods. We searched the literature to find all NIPT clinical validation and clinical experience studies between January 2011 and January 2022. Meta-analyses were performed using bivariate random-effects and univariate regression models for estimating summary performance measures across studies. Bivariate meta-regression was performed to explore the influence of testing method and study design. Subgroup and sensitivity analyses evaluated factors that may have led to heterogeneity. Based on 55 validation studies, the detection rate (DR) was significantly higher for retrospective studies, while the false positive rate (FPR) was significantly lower for prospective studies. Comparing the performance of NIPT methods for trisomies 21, 18, and 13 combined, the SNP method had a higher DR and lower FPR than other methods, significantly so for MPSS, though not for DANSR. The performance of the different methods in the 84 clinical experience studies was consistent with validation studies. Clinical positive predictive values of all NIPT methods improved over the last decade. We conclude that all NIPT methods are highly effective for fetal aneuploidy screening, with performance differences across methodologies.

Clinician-reported chorionicity and zygosity assignment using single-nucleotide polymorphism-based cell-free DNA: Lessons learned from 55,344 twin pregnancies.

OBJECTIVE: Prenatal chorionicity assessment relies on ultrasound, which can be confounded by many factors. Noninvasive assessment of zygosity is possible using single nucleotide polymorphism (SNP)-based cell-free DNA testing. Our objective was to determine the relationship between provider-reported chorionicity and SNP-cfDNA assignment of twin zygosity. METHODS: All twin pregnancy blood samples received by a reference laboratory between September 27, 2017 and September 8, 2021 were included. Chorionicity assignment was requested on the requisition, recorded as; monochorionic (MC), dichorionic, or "don't know". SNP-cfDNA zygosity results, monozygotic (MZ) or dizygotic (DZ), were correlated with chorionicity assignment. RESULTS: 59,471 twin samples (median gestational age = 12.0 weeks at draw) were received and analyzed; 55,344 (93.1%) received zygosity assignment. SNP-cfDNA reported 16,673 (30.1%) MZ and 38,671 (69.9%) as DZ. Provider-reported chorionicity was compared to the zygosity assignment for each case. Of 6283 provider-reported MC twins, 318 (5.1%) were reported as DZ using SNP-cfDNA. CONCLUSION(S): One in 20 suspected MC twin pregnancies were reported as DZ using SNP-cfDNA. Approximately 30% of 55,344 twin pregnancies were found to be MZ, including cases where chorionicity was unknown. SNP-cfDNA zygosity assessment is a useful adjunct assessment for twin pregnancies, particularly those reported as MC or without determined chorionicity.

Combining Donor-derived Cell-free DNA Fraction and Quantity to Detect Kidney Transplant Rejection Using Molecular Diagnoses and Histology as Confirmation.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) fraction and quantity have both been shown to be associated with allograft rejection. The present study compared the relative predictive power of each of these variables to the combination of the two, and developed an algorithm incorporating both variables to detect active rejection in renal allograft biopsies. METHODS: The first 426 sequential indication biopsy samples collected from the Trifecta study ( ClinicalTrials.gov # NCT04239703) with microarray-derived gene expression and dd-cfDNA results were included. After exclusions to simulate intended clinical use, 367 samples were analyzed. Biopsies were assessed using the molecular microscope diagnostic system and histology (Banff 2019). Logistic regression analysis examined whether combining dd-cfDNA fraction and quantity adds predictive value to either alone. The first 149 sequential samples were used to develop a two-threshold algorithm and the next 218 to validate the algorithm. RESULTS: In regression, the combination of dd-cfDNA fraction and quantity was found to be significantly more predictive than either variable alone ( P = 0.009 and P < 0.0001). In the test set, the area under the receiver operating characteristic curve of the two-variable system was 0.88, and performance of the two-threshold algorithm showed a sensitivity of 83.1% and specificity of 81.0% for molecular diagnoses and a sensitivity of 73.5% and specificity of 80.8% for histology diagnoses. CONCLUSIONS: This prospective, biopsy-matched, multisite dd-cfDNA study in kidney transplant patients found that the combination of dd-cfDNA fraction and quantity was more powerful than either dd-cfDNA fraction or quantity alone and validated a novel two-threshold algorithm incorporating both variables.

Association Between Total Cell Free DNA and SARS-CoV-2 In Kidney Transplant Patients: A Preliminary Study.

BACKGROUND: Kidney transplant (KT) recipients are at high risk for developing severe COVID-19. Lowering immunosuppression levels in KT recipients with COVID-19 encourages native immune responses but can raise the risk of rejection. Donor-derived cell-free DNA (dd-cfDNA), reported as a fraction of total cfDNA, is a proven biomarker for KT rejection. Total cfDNA levels are elevated in patients with COVID-19, which may depress dd-cfDNA fractions, potentially leading to missed rejections. METHODS: A retrospective analysis of 29 KT recipients hospitalized with COVID-19 between April and November 2020 examined total and dd-cfDNA levels. Blood samples were collected after onset of COVID-19, with follow-up samples collected from a subset of patients, when infection had likely subsided. RESULTS: After COVID-19 diagnosis, the median total cfDNA level was elevated (7.9 multiples of median [MoM]). A significant decrease in total cfDNA levels was observed between the first and second time points (6.2 MoM, 1.0 MoM; P <001). A significant positive association was identified between total cfDNA levels and COVID-19 severity (P = .02; R2 = .19). Two patients with biopsy-proven acute cellular rejection had dd-cfDNA fractions below the 1% cutoff for rejection (0.20% and 0.78%), with elevated total cfDNA levels of 7.9 MoM and 41.8 MoM, respectively. CONCLUSIONS: In this preliminary study, total cfDNA levels were elevated in KT patients with COVID-19, subsiding after resolution of infection. High total cfDNA levels may confound dd-cfDNA results, leading to failure to identify rejection. Considering total cfDNA levels is important in interpretation of dd-cfDNA tests for assessment of rejection in KT patients with COVID-19 or other infection.

A novel donor-derived cell-free DNA assay for the detection of acute rejection in heart transplantation.

BACKGROUND: Endomyocardial biopsy (EMB), the reference surveillance test for acute rejection (AR) in heart transplant (HTx) recipients, is invasive, costly, and shows significant interobserver variability. Recent studies indicate that donor-derived cell-free DNA (dd-cfDNA), obtained non-invasively from blood, is associated with AR and could reduce the frequency of EMB surveillance. The aim of this study was to examine the performance characteristics of a novel test for detecting AR in adult HTx recipients. METHODS: Plasma samples with contemporaneous EMBs were obtained from HTx recipients. A clinically available SNP-based massively multiplexed-PCR dd-cfDNA assay was used to measure dd-cfDNA fraction. dd-cfDNA fractions were compared with EMB-defined rejection status and test performance was assessed by constructing ROC curves and calculating accuracy measures. RESULTS: A total of 811 samples from 223 patients with dd-cfDNA testing and contemporaneous EMB were eligible for the study. dd-cfDNA fraction was significantly higher in AR (median 0.58%, IQR, 0.13%-1.68%) compared to non-AR (median 0.04%, IQR, 0.01%-0.11%, pc < 0.001). ROC analysis produced an area under the curve (AUC-ROC) of 0.86 (95% CI, 0.77-0.96). Defining samples with dd-cfDNA fraction ≥0.15% as AR yielded 78.5% sensitivity (95% CI, 60.7%-96.3%) and 76.9% specificity (95% CI, 71.1%-82.7%). Positive and negative predictive values were 25.1% (95% CI, 18.8%-31.5%) and 97.3% (95% CI, 95.1%-99.5%) respectively, calculated using the cohort AR prevalence of 9.0% (95% CI, 5.3%-12.8%) with adjustment for repeat samples. CONCLUSIONS: This novel dd-cfDNA test detects AR in HTx recipients with good accuracy and holds promise as a noninvasive test for AR in HTx recipients.

Non-invasive prenatal screening for fetal triploidy using single nucleotide polymorphism-based testing: Differential diagnosis and clinical management in cases showing an extra haplotype.

OBJECTIVE: An extra haplotype is infrequently encountered in single nucleotide polymorphism(SNP)-based non-invasive prenatal testing (NIPT) and is usually attributed to an undetected twin or triploidy. We reviewed a large series to establish relative frequencies of these outcomes and identify alternative causes. METHODS: In 515,804 women receiving NIPT from September 2017 through March 2019, all results with an extra haplotype were reviewed. Known viable and vanished twin pregnancies were excluded. For positive cases, pregnancy outcome information was sought. RESULTS: Of 1005 results with an extra haplotype (1 in 513), pregnancy outcome was available for 773 cases: 11% were confirmed or suspected triploidy; 65% to vanished twin; 10% with pregnancy loss. Rare explanations included complete mole, chimera, undisclosed donor egg pregnancy, maternal organ transplant and one instance of maternal neoplasm. Among triploid cases that were detected and independently confirmed, 23/27 (85%) were diandric. CONCLUSION: SNP-based NIPT, with detection of an extra haplotype, is 11% predictive of triploidy. For results with an extra haplotype, ultrasound is recommended to establish viability, evaluate for twins (viable or vanished), and detect findings consistent with triploidy. Review of patient history, serum screening, and ultrasound will reduce the number of CVS or amniocenteses necessary to confirm a diagnosis of triploidy.

Clinical Validation of a Plasma Donor-derived Cell-free DNA Assay to Detect Allograft Rejection and Injury in Lung Transplant.

Background: Lung transplant patients are vulnerable to various forms of allograft injury, whether from acute rejection (AR) (encompassing acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), chronic lung allograft dysfunction (CLAD), or infection (INFXN). Previous research indicates that donor-derived cell-free DNA (dd-cfDNA) is a promising noninvasive biomarker for the detection of AR and allograft injury. Our aim was to validate a clinical plasma dd-cfDNA assay for detection of AR and other allograft injury and to confirm and expand on dd-cfDNA and allograft injury associations observed in previous studies. Methods: We measured dd-cfDNA fraction using a novel single-nucleotide polymorphism-based assay in prospectively collected plasma samples paired with clinical-pathologic diagnoses. dd-cfDNA fraction was compared across clinical-pathologic cohorts: stable, ACR, AMR, isolated lymphocytic bronchiolitis, CLAD/neutrophilic-responsive allograft dysfunction (NRAD), and INFXN. Performance characteristics were calculated for AR and combined allograft injury (AR + CLAD/NRAD + INFXN) versus the stable cohort. Results: The study included 195 samples from 103 patients. Median dd-cfDNA fraction was significantly higher for ACR (1.43%, interquartile range [IQR]: 0.67%-2.32%, P = 5 × 10-6), AMR (2.50%, IQR: 2.06%-3.79%, P = 2 × 10-5), INFXN (0.74%, IQR: 0.46%-1.38%, P = 0.02), and CLAD/NRAD (1.60%, IQR: 0.57%-2.60%, P = 1.4 × 10-4) versus the stable cohort. Area under the receiver operator characteristic curve for AR versus stable was 0.91 (95% confidence interval [CI]: 0.83-0.98). Using a ≥1% dd-cfDNA fraction threshold, sensitivity for AR was 89.1% (95% CI: 76.2%-100.0%), specificity 82.9% (95% CI: 73.3%-92.4%), positive predictive value, 51.9% (95% CI: 37.5%-66.3%), and negative predictive value, 97.3% (95% CI: 94.3%-100%). For combined allograft injury area under the receiver operator characteristic curve was 0.76 (95% CI: 0.66-0.85), sensitivity 59.9% (95% CI: 46.0%-73.9%), specificity 83.9% (95% CI: 74.1%-93.7%), positive predictive value, 43.6% (95% CI: 27.6%-59.6%), and negative predictive value, 91.0% (95% CI: 87.9%-94.0%). Conclusions: These results indicate that our dd-cfDNA assay detects AR and other allograft injury. dd-cfDNA monitoring, accompanied by standard clinical assessments, represents a valuable precision tool to support lung transplant health and is appropriate for further assessment in a prospective randomized-controlled study.

Impact of Circulating Tumor DNA-Based Detection of Molecular Residual Disease on the Conduct and Design of Clinical Trials for Solid Tumors.

PURPOSE: Earlier detection of cancer recurrence using circulating tumor DNA (ctDNA) to detect molecular residual disease (MRD) has the potential to dramatically affect cancer management. We review evidence supporting the use of ctDNA as a biomarker for detection of MRD and highlight the potential impact that ctDNA testing could have on the conduct of clinical trials. METHODS: We searched the literature using MEDLINE (via PubMed) for articles from January 1, 2000, focusing on studies that assessed ctDNA as a predictor of cancer recurrence. Broadly focused searches on ctDNA and cancer were also performed to provide additional background information. www.clinialtrials.gov was searched to identify trials that incorporate ctDNA testing. RESULTS: Numerous studies across different cancer types indicate that ctDNA-based MRD detection predicts recurrence with high sensitivity and specificity, and with lead times that precede standard imaging by up to 12 months. Recently, ctDNA testing has started being used to enroll MRD-positive patients at high risk of recurrence into trials, promising gains in statistical power that allow clinical utility to be demonstrated with smaller cohorts. Trials where ctDNA testing based-MRD detection is used to stratify patients into low or high-risk categories for treatment assignment are also ongoing. In addition, there is increasing evidence supporting the use of ctDNA dynamics or clearance as a surrogate end point, which could significantly reduce trial duration. CONCLUSION: ctDNA-based trial enrichment across many cancers seems likely to become increasingly common for cost- and time-reduction benefits. Trial efficiency could also benefit from using ctDNA as a surrogate end point, leading to accelerated approval of new therapeutics. A clear demonstration of efficacy from trials that use ctDNA-based MRD detection to assign treatment could transform clinical practice.