Fraction Of Cell-free DNA Research Articles

Fetal Fraction of Cell-Free DNA in the Prediction of Adverse Pregnancy Outcomes: A Nationwide Retrospective Cohort Study.

To assess the added value of fetal fraction of cell-free DNA in the maternal circulation in the prediction of adverse pregnancy outcomes. Retrospective cohort study. Nationwide implementation study on non-invasive prenatal testing (NIPT; TRIDENT-2 study). Pregnant women in the Netherlands opting for NIPT between June 2018 and June 2019. Two logistic regression prediction models were constructed for each adverse pregnancy outcome. The first model (base model) included prognostic clinical parameters that were selected from existing first-trimester prediction models for adverse pregnancy outcomes. The second model (fetal fraction model) included fetal fraction as a predictor on top of the prognostic clinical parameters included in the base model. The added prognostic value of fetal fraction was assessed by comparing the base and fetal fraction models in terms of goodness of fit and predictive performance. Likelihood ratio test (LRT), area under the curve (AUC) and Integrated Discrimination Improvement (IDI) index. The study cohort consisted of 56 110 pregnancies. The incidence of adverse pregnancy outcomes was 5.7% for hypertensive disorders of pregnancy (HDP; n = 3207), 10.2% for birthweight < p10 (n = 5726), 3.2% for birthweight < p2.3 (n = 1796), 3.4% for spontaneous preterm birth (sPTB; n = 1891), 3.4% for diabetes (n = 1902) and 1.3% for congenital anomalies (n = 741). Adding fetal fraction to the base model improved model fit for HDP, birthweight < p10, birthweight < p2.3, all sPTB, and diabetes, but not for congenital anomalies (LRT p < 0.05). For HDP, the AUC improved from 0.67 to 0.68 by adding fetal fraction to the base model (p = 0.14) with an IDI of 0.0018 (p < 0.0001). For birthweight < p10, the AUC improved from 0.65 to 0.66 (p < 0.0001) with an IDI of 0.0023 (p < 0.0001). For birthweight < p2.3, the AUC improved from 0.67 to 0.69 (p < 0.0001) with an IDI of 0.0011 (p < 0.0001). For all sPTB, the AUC was similar for both models (AUC 0.63, p = 0.021) with an IDI of 0.00028 (p = 0.0023). For diabetes, the AUC was similar (AUC 0.72, p = 0.35) with an IDI of 0.00055 (p = 0.00015). Fetal fraction has statistically significant but limited prognostic value in the prediction of adverse pregnancy outcomes in addition to known prognostic clinical parameters.

V-212.2: Enhancing pediatric kidney transplant rejection detection: Integrating donor-derived cell-free DNA fraction and quantity.

V-212.2: Enhancing pediatric kidney transplant rejection detection: Integrating donor-derived cell-free DNA fraction and quantity.

Bridging horizons beyond CIRCULATE-Japan: a new paradigm in molecular residual disease detection via whole genome sequencing-based circulating tumor DNA assay.

Circulating tumor DNA (ctDNA) is the fraction of cell-free DNA in patient blood that originates from a tumor. Advances in DNA sequencing technologies and our understanding of the molecular biology of tumors have increased interest in exploiting ctDNA to facilitate detection of molecular residual disease (MRD). Analysis of ctDNA as a promising MRD biomarker of solid malignancies has a central role in precision medicine initiatives exemplified by our CIRCULATE-Japan project involving patients with resectable colorectal cancer. Notably, the project underscores the prognostic significance of the ctDNA status at 4weeks post-surgery and its correlation to adjuvant therapy efficacy at interim analysis. This substantiates the hypothesis that MRD is a critical prognostic indicator of relapse in patients with colorectal cancer. Despite remarkable advancements, challenges endure, primarily attributable to the exceedingly low ctDNA concentration in peripheral blood, particularly in scenarios involving low tumor shedding and the intrinsic error rates of current sequencing technologies. These complications necessitate more sensitive and sophisticated assays to verify the clinical utility of MRD across all solid tumors. Whole genome sequencing (WGS)-based tumor-informed MRD assays have recently demonstrated the ability to detect ctDNA in the parts-per-million range. This review delineates the current landscape of MRD assays, highlighting WGS-based approaches as the forefront technique in ctDNA analysis. Additionally, it introduces our upcoming endeavor, WGS-based pan-cancer MRD detection via ctDNA, in our forthcoming project, SCRUM-Japan MONSTAR-SCREEN-3.

Abstract 406: Characterization of whole genome duplication in a genomic cohort of over 14000 cell free DNA samples

Abstract Background: Wide-scale rates of whole genome duplication (WGD) have previously been demonstrated in studies performed in tissue among genomically characterized tumors. Such studies report varied rates of WGD depending on tumor type and stage. Hallmarks of WGD include widespread loss-of-heterozygosity as well as associations with tumor proliferation and specific oncogenic driver events such as loss of function TP53 mutations. It has been proposed that presence of WGD could be a poor prognostic biomarker, leading to interest in profiling this genomic landmark in large retrospective cohorts to inform future clinical studies. Methods: Samples that were run through the GuardantOMNI high content genomic profiling assay using cfDNA extracted from blood were analyzed. Only samples originating from individuals with primary breast, colorectal, or prostate cancer were considered due to limited sample size in other indications. We deployed a likelihood-based copy-number variant caller, which jointly selects tumor purity and ploidy while fitting normalized coverage and germline variant allele frequencies across genome-wide data. Only samples where we predicted to have greater than 10% tumor fraction from copy number variant (CNV) profiles were considered for statistical analysis. The presence of whole genome duplication was assessed by counting the median major chromosomal copy number. Presence of SNV/InDels were annotated, assessed for pathogenicity, and filtered to those with a pathogenic allele frequency greater than 10%. Results: A total of 14,076 samples were analyzed, with 5362 passing the 10% observed tumor fraction cutoff. WGD was annotated in 2195 (41%) samples with varying rates across sub-cohorts (31%, 36%, and 54% of breast, colorectal, and prostate cancer, respectively). Mutation in TP53 was associated with a large increase in rate of WGD (odds ratio 6.9, 95% CI 6.1-7.8) which is consistent but higher in magnitude to similar studies in tissue samples. Amplification of CCNE1 and homozygous deletion of genes including WRN and RB1 were also associated with WGD, independent of TP53 mutation status and overall rates of aneuploidy. Conclusion: WGD was detected in 41% of samples with greater than 10% tumor fraction in cell-free DNA across three cancer types. While this represents a substantial percentage of advanced cancers, more research needs to be done regarding the impact of these events on clinical outcomes and treatment response. Additionally, co-occurrence of these events with other advanced cancer biomarkers such as microsatellite instability or homologous recombination deficiency was not reported. Given the high rates of these events, future retrospective studies are warranted to correlate presence of WGD with patient outcomes and response. Citation Format: Andrew Gross, Hao Wang, Denis Tolkunov, Colby Jenkins, Sara Wienke, Catalin Barbacioru, Han-Yu Chuang. Characterization of whole genome duplication in a genomic cohort of over 14000 cell free DNA samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 406.

Association between fetal fraction of cell-free DNA and adverse pregnancy outcomes.

To determine the association between fetal fraction (FF) levels in cell-free fetal DNA (cffDNA) testing and adverse pregnancy outcomes. This retrospective cohort study, conducted at a single center, involved 2063 pregnant women with normal 1st and 2nd trimester non-invasive prenatal test (NIPT) results between 2016 and 2021. Pregnancy outcomes were examined by determining the < 4% and < 5th percentile (3.6%) cut-off values for low fetal fraction (LFF). Pregnancy outcomes were also examined by dividing the FF into population-based quartiles. Adverse pregnancy outcomes were pregnancy-induced hypertensive diseases (PIHD), gestational diabetes mellitus (GDM), spontaneous preterm birth (PTB), intrahepatic cholestasis of pregnancy (ICP), small for gestational age (SGA), large for gestational age (LGA), low birth weight (LBW), macrosomia, and 1st and 5th minutes low APGAR scores (< 7). PIHD was significantly higher in LFF (< 4% and < 5th percentile) cases (p = 0.015 and p < 0.001, respectively). However, in population-based quartiles of FF, PIHD did not differ significantly between groups. Composite adverse maternal outcomes were significantly higher in the FF < 4% group (p = 0.042). When analyzes were adjusted for maternal age, BMI, and gestational age at NIPT, significance was maintained at < 4%, < 5th percentile LFF for PIHD, and < 4% LFF for composite adverse maternal outcomes. However, there was no significant relationship between LFF with GDM, ICP and PTB. Additionally, there was no significant association between low APGAR scores, SGA, LGA, LBW, macrosomia, and LFF concerning neonatal outcomes. Our study showed that LFF in pregnant women with normal NIPT results may be a predictor of subsequent PIHD.

Fetal fraction of cell-free DNA in noninvasive prenatal testing and adverse pregnancy outcomes: a nationwide retrospective cohort study of 56,110 pregnant women

BackgroundNon-invasive prenatal testing by cell-free DNA analysis is offered to pregnant women worldwide to screen for fetal aneuploidies. In non-invasive prenatal testing, the fetal fraction of cell-free DNA in the maternal circulation is measured as a quality control parameter. Since fetal cell-free DNA originates from the placenta, the fetal fraction might also reflect placental health and maternal pregnancy adaptation. ObjectiveTo assess the association between the fetal fraction and adverse pregnancy outcomes. Study DesignWe performed a retrospective cohort study of women with singleton pregnancies opting for non-invasive prenatal testing between June 2018 and June 2019 within the Dutch nationwide implementation study (TRIDENT-2). Multivariable logistic regression analysis was used to assess associations between fetal fraction and adverse pregnancy outcomes. Fetal fraction was assessed as a continuous variable and as <10th percentile, corresponding to a fetal fraction below 2.5%. ResultsThe cohort comprised 56,110 pregnancies. In the analysis of fetal fraction as a continuous variable, a decrease in fetal fraction was associated with an increased risk of hypertensive disorders of pregnancy (adjusted OR [aOR] 2.27 [95% CI 1.89 to 2.78]), small for gestational age neonates <p10 (aOR 1.37 [1.28 to 1.45]) and <p2.3 (aOR 2.63 [1.96 to 3.57]), and spontaneous preterm birth from 24 to 37 weeks of gestation (aOR 1.02 [1.01 to 1.03]). No association was found for fetal congenital anomalies (aOR 1.02 [1.00 to 1.04]), stillbirth (aOR 1.02 [0.96 to 1.08]), or neonatal death (aOR 1.02 [0.96 to 1.08]). Similar associations were found for adverse pregnancy outcomes when fetal fraction was <10th percentile. ConclusionsIn early pregnancy, a low fetal fraction is associated with an increased risk of adverse pregnancy outcomes. These findings can be utilized to expand the potential of non-invasive prenatal testing in the future, enabling the prediction of pregnancy complications and facilitating tailored pregnancy management through intensified monitoring or preventive measures.

Association between the first and second trimester cell free DNA fetal fraction and spontaneous preterm birth

ABSTRACT Objective To evaluate whether the fetal fraction of cell-free DNA at the first and second trimesters is associated with spontaneous preterm birth. Methods This was a retrospective cohort study with singleton pregnancies who underwent noninvasive prenatal testing. According to pregnancy outcome, eligible patients were divided into a delivery group ≥37 weeks of pregnancy (term group) and <37 weeks of pregnancy (spontaneous preterm group). Stepwise linear regression was used to identify maternal characteristics associated with the fetal fraction of cell‐free DNA. Logistic regression analysis was performed to evaluate the association between the fetal fraction of cell-free DNA and spontaneous preterm birth, adjusted for confounding factors. Results 14,020 cases were included in the study, 13292 cases (94.81%) in the term group and 728 cases (5.19%) in the spontaneous preterm group. The cell-free fraction of fetal DNA was inversely correlated with maternal age and body mass index. Positively correlated with gestational age, fertility, and assisted reproductive technology. After adjusting for the covariates, logistic regression analysis revealed no statistically significant association between the fetal fraction of cell-free DNA and spontaneous preterm birth. Conclusion In our original study, we found no association between the fetal fraction on NIPT and subsequent spontaneous preterm birth.

(996) Relationship Between Absolute Quantification of Donor-Derived Cell-Free DNA and Donor-Derived Cell-Free DNA Fraction for Detection of Allograft Rejection in Heart Transplant Patients

(996) Relationship Between Absolute Quantification of Donor-Derived Cell-Free DNA and Donor-Derived Cell-Free DNA Fraction for Detection of Allograft Rejection in Heart Transplant Patients

Correlation of low fetal fraction of cell-free DNA at the early second-trimester and pregnancy complications related to placental dysfunction in twin pregnancy.

This study aimed to determine the correlation between fetal fraction (FF) of cell-free DNA (cf-DNA) and pregnancy complications related to placental dysfunction in Twin Pregnancy. This retrospective cohort study analyzed twin pregnant women who underwent non-invasive prenatal testing (NIPT) at 12+0-26+6 weeks of gestation from April 2017 to April 2021. Low fetal fraction (LFF) was defined individually as less than the 25th, 10th, 5th, and 2.5th percentile among all fetal fractions in the cohort. Primary outcomes included gestational hypertension (GH), preeclampsia (PE), gestational diabetes mellitus (GDM), and small for gestational age (SGA). Logistic regression analysis was used to assess the relationship between LFF and pregnancy complications. A total of 500 twin pregnancies (male-male twins, 245; female-female twins, 255) were included in this study. In LFF group (FF < 25th percentiles), maternal BMI was significantly higher than FF > 75th percentiles (23.6 kg/m2 vs. 21.3 kg/m2; P < 0.001). The risk of SGA increased gradually from FF < 25th percentiles [adjusted odds ratio (OR), 1.71; 95% confidence interval (CI), 1.07-2.99; P = 0.016] to FF < 2.5th percentiles (adjusted OR, 4.44; 95% CI,1.33-14.82; P < 0.015). In addition, the risks of SGA in both fetuses were higher than the risks of at least one fetus SGA in LFF group. LFF had no correlation with GH, PE, and GDM in twin pregnancy. LFF has a strong association with increased risk of SGA in twin pregnancy. Moreover, FF of cf-DNA may provide a new idea for the early screening of diseases related to placental dysfunction in twin pregnancy.

The effect of abnormal placentation on maternal serum fetal fraction of cell-free DNA.

Abnormal placentation may affect the maternal serum fraction of cell-free fetal DNA (fetal fraction) determined as part of non-invasive prenatal screening (NIPS). This study aimed to assess whether the fetal fraction can predict placenta accreta spectrum (PAS) with or without placenta previa (PP). We also investigated the impact of trophoblastic invasion depth on the fetal fraction. This is a retrospective case-control study of pregnant women with and without abnormal placentation carrying a singleton and having undergone NIPS prior to 20weeks of gestation. The eligible subjects were selected from a cohort managed at our institution for PAS suspected antenatally. We compared women with normal placentation (controls) to PAS, PP, or PAS+PP cases. Data were abstracted from electronic medical records, and PAS was confirmed histologically. Of the 146 patients in our cohort, 8 controls, 10 PP, 6 PAS, and 7 PAS+PP cases were eligible for the study. Among the groups, there were no significant differences in baseline demographic and clinical characteristics except the median number of prior uterine surgeries. Also, the groups did not significantly differ in their median fetal fraction. The fetal fraction did not discriminate any group when stratified according to the depth of placental invasion, i.e.,no PAS, abnormally adherent, and abnormally invasive placenta. The maternal serum fraction of cell-free fetal DNA measured before 20weeks of gestation is not predictive of PAS with or without concurrent PP or the depth of trophoblastic invasion.

Low Fetal Fraction of Cell Free DNA at Non-Invasive Prenatal Screening Increases the Subsequent Risk of Preterm Birth in Uncomplicated Singleton Pregnancy.

ObjectiveTo examine the association between low fetal fraction (FF) of cell free DNA determined at non-invasive prenatal screening (NIPS) and the subsequent risk of preterm birth in uncomplicated singleton pregnancy.MethodsWe retrospectively interrogated NIPS System and hospitalization records from April 2018 to August 2019 and obtained results from 1521 consecutive and uncomplicated women with singleton pregnancy in which plasma FF of cell free DNA at NIPS had been investigated together with birth outcomes. We examined the association between FF and preterm birth (PTB) by regression analysis.ResultsThe incidence of preterm birth, low birthweight, and macrosomia in the study population was 5.06%, 2.89%, and 7.17%, respectively. FF at NIPS in the second to fourth quartiles (8.40–11.07, 11.08–13.70, and >13.70%, respectively) was associated with higher gestational age at delivery relative to the lowest quartile (<8.40%), with estimated mean increases of 0.27 weeks (95% CI: 0.05–0.49), 0.29 weeks (95% CI: 0.06–0.51), and 0.28 weeks (95% CI: 0.05–0.51), respectively (P for trend = 0.027). Low FF (< the 5th percentile) was associated with an increased risk of PTB (adjusted OR: 2.23, 95% CI: 1.01–4.98, P = 0.047) compared to normal FF (≥ the 5th and ≤ the 95th percentiles). In addition, when compared to women with normal FF and body mass index (BMI) <25 at NIPS, the risk of early PTB (< 34 weeks gestation) was remarkably significantly higher among those with low FF and BMI ≥25 (adjusted OR: 6.29, 95% CI: 1.71–23.15, P = 0.006).ConclusionOur study supports the association of low FF at NIPS with PTB (especially early PTB) for uncomplicated singleton pregnancy.

Abstract 3745: Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression

Abstract Reduction of global DNA methylation is a characteristic epigenetic alteration of various cancer types, including colorectal cancer. Abnormality of several factors, such as DNA methyltransferases (DNMT), demethylases, or deviation in methyl-donor (folate and S-adenosylmethionine) availability can contribute to the development of genome-wide hypomethylation. Detection of epigenetic changes as global DNA hypomethylation in cell-free DNA fraction obtained from blood samples can expand the opportunities for the early recognition of colorectal cancer. One of our main goals was the investigation of global DNA methylation patterns in tissue biopsies (n=183) and cell-free DNA fraction of blood samples (n=48) along the colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore possible underlying mechanisms of genome-wide hypomethylation formation in 12 colorectal tumor tissue sections, containing transition zones. Using LINE-1 pyrosequencing, significantly reduced global DNA methylation level was detected in line with cancer progression in tissue specimens (normal: 77.5±1.7%, adenoma: 72.7±4.8%, carcinoma: 69.7±7.6%, p≤0.0001) and in liquid biopsies as well (normal: 82.0±2.0%, adenoma: 80.0±1.7%, carcinoma: 79.8±1.3%, p≤0.01). However, no significant methylation changes were found in inflammatory bowel disease cases. Analyzing microarray data in silico, altered mRNA expression of certain methylation-, and one-carbon metabolism-related genes were detected in tumorous specimens vs. healthy biopsies, from which DNMT1 was upregulated, and folate receptor 2 (FOLR2) was downregulated. DNMT and FOLR2 expression were validated by immunohistochemistry. Furthermore, significantly reduced folic acid and S-adenosylmethionine content were observed in parallel with diminishing 5-methylcytosine levels in adenoma and carcinoma sections compared to normal adjacent to tumor tissue areas by immunolabeling (p≤0.05). Our results suggest that intraindividual monitoring of genome-wide hypomethylation may assist in the recognition of adenoma formation, cancer progression, or remission as well. Moreover, lower global DNA methylation level could be connected to decreased methyl-donor availability with the contribution of reduced FOLR2 expression. Citation Format: Krisztina Andrea Szigeti, Alexandra Kalmár, Gábor Valcz, Barbara Kinga Barták, Zsófia Nagy, Sára Zsigrai, Ildikó Felletár, Árpád V Patai, Tamás Micsik, Márton Papp, Eszter Márkus, Zsolt Tulassay, Péter Igaz, István Takács, Béla Molnár. Global DNA hypomethylation can be linked to decreased methyl-donor content in colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3745.

Comparison of the Fetal Fraction of Cell-Free DNA in In-Vitro Fertilization (IVF) Versus Natural Conception Evaluation of the Fetal Fraction With IVF Parameters.

BackgroundAs the offspring of assisted reproduction techniques (ARTs) have become a substantial proportion of the population, increased attention has been placed on the safety of ART. Investigators have focused on identifying a tool that combines molecular or biological tests that can predict the outcomes of in-vitro fertilization (IVF) or intracytoplasmic sperm injection and the resulting pregnancy after ART-mediated embryo implantation. This study aimed to answer the following questions: is there a difference between natural conception and IVF pregnancies regarding fetal fraction (FF) of cell-free DNA (cfDNA) in maternal age, birth weight, gender, and gestational age? Is there a difference between FF concentration regarding the parameters of IVF as possible predictive factors affecting the outcomes of IVF?MethodologyThis study included 31 women with singleton pregnancies conceived via IVF who underwent cell-free fetal DNA (cffDNA) screening for trisomy 13, 18, and 21; sex determination; and FF. The control group included 55 women who experienced natural conception. For all women, anthropometric characteristics such as age, weight, height, and body mass index (BMI) were recorded. For the IVF group, early follicular phase values of follicle-stimulating hormone, luteinizing hormone, prolactin, anti-müllerian hormone, thyroid-stimulating hormone, and estradiol were recorded.ResultsThe natural conception and IVF groups were similar regarding maternal age, BMI of the mother, gender, birth weight, and gestational age. FF was not significantly different between the natural conception and IVF groups (10 (3.8) vs. 9 (2.6); p = 0.144). The results were similar after adjusting for maternal age via regression analysis. cfDNA was not associated with maternal age, birth weight, gender, or gestational age in the entire study sample or separately for the natural conception and IVF groups. No significant correlation was found between cfDNA and IVF parameters.ConclusionsThe FF is an important factor for non-invasive prenatal testing (NIPT) accuracy. Several studies have found a reduction in FF in pregnancies following ART compared with natural conception, while other studies have presented no differences in the FF. All researchers agree on the importance of NIPT; however, knowledge on how the FF is affected in ART pregnancies compared with naturally conceived pregnancies is very limited. In this study, no difference in FF for the IVF group compared with natural conception women was observed. The cffDNA concentrations in maternal serum do not appear to be affected in IVF conception. We suggest that FF is an independent factor compared with IVF parameters.

Quantitative Donor-Derived Cell-Free DNA Levels Reflect the Variability in Lung Allograft Cellular Injury

<h3>Purpose</h3> Recently we clinically validated a donor-derived cell-free DNA fraction (%dd-cfDNA) test for detecting acute rejection (AR) in lung transplant recipients (LTR). The sensitivity and specificity to detect AR was 89.1% and 82.9% respectively. We now assess the clinical validity of using quantitative absolute dd-cfDNA levels for detecting AR and other graft injury. <h3>Methods</h3> We measured %dd-cfDNA and absolute dd-cfDNA (cp/mL) in 195 prospectively collected plasma samples paired with clinical-pathologic diagnoses using a massively-multiplexed PCR SNP-based assay (Prospera™, Natera, Inc.). Absolute dd-cfDNA levels were compared for the clinical-pathologic cohorts: acute cellular rejection (ACR), antibody-mediated rejection (AMR), chronic lung allograft dysfunction or neutrophilic-responsive allograft dysfunction (CLAD/NRAD), infection (INFXN), and STABLE (no graft injury). Performance characteristics were calculated for AR (ACR+AMR) vs STABLE. Linear regression analysis compared absolute dd-cfDNA to %dd-cfDNA. <h3>Results</h3> Absolute dd-cfDNA levels were significantly higher for injury cohorts, AR, and combined allograft injury (AR+CLAD/NRAD+INFXN) vs STABLE (TABLE1). Using absolute dd-cfDNA to distinguish AR from STABLE, the area under the receiver operating characteristic curve was 0.87 (95% CI:0.78-0.95). The associated sensitivity and specificity was 79.5% (95% CI:71.3-96.6) and 80.3% (95% CI:70.2-90.4), using a 90 cp/mL threshold. There was a strong association between absolute dd-cfDNA and %dd-cfDNA for STABLE (R²=0.74, p=9.6 × 10⁻⁵⁹) and combined allograft injury (R²=0.61, p=6.5 × 10⁻³⁷). <h3>Conclusion</h3> Absolute dd-cfDNA levels have clinical validity for detecting AR and other forms of allograft injury and are strongly associated with %dd-cfDNA. Further research will investigate whether absolute dd-cfDNA measurement offers additional precision for allograft cellular injury assessment and longitudinal responses to therapy beyond %dd-cfDNA.

PEAC: An Ultrasensitive and Cost-Effective MRD Detection System in Non-small Cell Lung Cancer Using Plasma Specimen.

Circulating tumor DNA (ctDNA), a tumor-derived fraction of cell-free DNA (cfDNA), has emerged as a promising marker in targeted therapy, immunotherapy, and minimal residual disease (MRD) monitoring in postsurgical patients. However, ctDNA level in early-stage cancers and postsurgical patients is very low, which posed many technical challenges to improve the detection rate and sensitivity, especially in the clinical practice of MRD detection. These challenges usually include insufficient DNA input amount, limit of detection (LOD), and high experimental costs. To resolve these challenges, we developed an ultrasensitive ctDNA MRD detection system in this study, namely PErsonalized Analysis of Cancer (PEAC), to simultaneously detect up to 37 mutations, which account for 70–80% non-small cell lung cancer (NSCLC) driver mutations from low plasma sample volume and enables LOD of 0.01% at a single-site level. We demonstrated the high performance achieved by PEAC on both cfDNA reference standards and clinical plasma samples from three NSCLC patient cohorts. For cfDNA reference standards, PEAC achieved a specificity of 99% and a sensitivity of 87% for the mutations at 0.01% allele fraction. In the second cohort, PEAC showed 100% concordance rate between ddPCR and Next-generation sequencing (NGS) among 29 samples. In the third cohort, 22 of 59 patients received EGFR TKI treatment. Among them, three in four patients identified low level actionable gene mutations only by PEAC had partial responses after targeted therapy, demonstrating high ctDNA detection ability of PEAC. Overall, the developed PEAC system can detect the majority of NSCLC driver mutations using 8–10 ml plasma samples, and has the advantages of high detection sensitivity and lower costs compared with the existing technologies such as ddPCR and NGS. These advantages make the PEAC system quite appropriate for ctDNA and MRD detection in early-stage NSCLC and postsurgical recurrence monitoring.

EP443: Fetal fraction amplification within NIPS enables detection of clinically-relevant genome-wide copy-number variants to 1Mb resolution

While clinically relevant copy number variants (CNVs) occur in 6-8% of pregnancies with fetal structural anomalies, they are typically difficult to detect via noninvasive prenatal screening (NIPS) because of the low fetal fraction in cell free DNA (cfDNA) isolated from maternal plasma. By leveraging fetal fraction amplification, a method to preferentially sequence shorter fragments that are enriched for fetal-derived cfDNA, we can detect small CNVs with high sensitivity. Here we describe an analytical validation of an NIPS that can detect clinically relevant CNVs across the genome.

Abstract P2-07-01: Blood tumor mutational burden (bTMB) and blood copy number burden (bCNB) by genome-wide circulating tumor DNA (ctDNA) assessment predict outcome and resistance in hormone-receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients (pts) treated with CDK4/6 inhibitor (CDK4/6i)

Abstract Background: CDK4/6i combined with ET improves survival for pts with HR+, HER2- MBC. However, biomarkers to predict efficacy and resistance are needed. We hypothesized that a comprehensive next-generation sequencing (NGS)-based liquid biopsy assessment of ctDNA mutation and copy number analysis may identify novel prognostic and predictive biomarkers. Methods: We collected serial blood for ctDNA testing from the 51 pts with HR+, HER2- MBC enrolled in the Alt Dose Palbo trial (NCT03007979), a single-arm phase II study of palbociclib plus letrozole or fulvestrant at 5 days on/2 days off weekly schedule as the first- or second-line ET. The median follow up was 38.2 months at data cutoff for this analysis. Plasma collected at baseline (BL) from all 51 pts, at progression (PD) from all 20 pts who have progressed, and at additional interim timepoints from 2 pts to explore longitutinal changes, with paired germline DNA, were subjected to the PredicineWES+ assay and low-pass whole-genome sequencing (lpWGS). The PredicineWES+ assay provides deep sequencing of 600 genes in the PredicineATLAS panel combined with whole-exome sequencing (WES) of all exonic regions of 20,000 genes enabling genome-wide detection of somatic single nucleotide variation (SNV), indels, copy number variation (CNV) and determination of bTMB. LpWGS offers an unbiased and high-throughput assessment of CNVs and tumor fraction in cell free DNA to derive bCNB. Statistical associations of bTMB, bCNB, and individual alterations with clinical benefit (CB), defined as no PD at 24 weeks by RECIST 1.1, and progression-free survival (PFS) were examined by Fisher’s exact test, Kaplan-Meier analysis, and Cox model. P values were adjusted to control false discovery rate (FDR). Results: The BL median bTMB was 1.6 mutations per megabase pair [IQR 0.6-3.5]. Pts with CB (N=41, 80%) had significantly lower bTMB scores (median [IQR] 1.2 [0.6-2.6] vs. 8.3 [2.4-21.4], (P&amp;lt;0.01) and significantly lower bCNB (P=0.03). bTMB and bCNB were highly correlated (Spearman correlation ρ =0.77, P=6.2E-11). bTMB above the predefined cutoff of 16 identified pts with significantly shorter PFS (4.7 vs 18.2 months, HR 3.73, CI 1.24-11.27, P=0.01). In contrast, BL clinical features including ET sensitivity, bone only vs. visceral, or de novo vs. recurrent MBC, were not associated with CB. BL alterations (SNV or CNV) in 91 genes, occurring in at least 3 pts each, were significantly associated with worse PFS (adjusted P&amp;lt;0.05), 61 of which were outside of the 600-gene panel. The 91 genes included both novel and previously reported alterations implicated in CDK4/6i and ET resistance. Examples include AR, ATM, AURKA, BRCA2, CCND1, DDR2, ESR1, FAT1, FGFR4, FOXP1, MYC, RB1, and RUNX1T1, some of which were also enriched at PD. Additional novel variants were enriched at PD, including ABCC12, ABCA13, SHANK1, and TET1. Serial analysis at BL, cycle 1 day 15 (C1D15), C2D1, the last Q3-month staging scan without PD, and at imaging detection of PD from 2 pts revealed a decline in bCNB at C1D15, then plateau, followed by an increase in bCNB that preceded imaging detection of PD. Conclusion: The comprehensive ctDNA profiling approach with PredicineWES+ and lpWGS identified high BL bTMB and bCNB as poor prognostic biomarkers in pts on CDK4/6i and ET. Our study also discovered novel candidate genes involved in cell cycle and DNA damage repair pathways in association with poor outcome. Results from this study highlight the genomic heterogeneity of HR+, HER2- MBC and provide important insights in the understanding of CDK4/6i and ET resistance mechanisms to guide therapeutic development in pts with resistant disease. Citation Format: Andrew Davis, Jingqin Luo, Tiantian Zheng, Lu Tan, Amy Wang, Rama Suresh, Foluso Ademuyiwa, Caron Rigden, Timothy Rearden, Katherine Clifton, Katherine Weilbaecher, Ashley Frith, Pavan K Tandra, Tracy Summa, Brittney Haas, Shana Thomas, Leonel Hernandez-Aya, Lindsay Peterson, Shujun Luo, Bonnie L King, Shidong Jia, Jianjun Yu, Pan Du, Jairam Krishnamurthy, Cynthia X Ma, C Dai. Blood tumor mutational burden (bTMB) and blood copy number burden (bCNB) by genome-wide circulating tumor DNA (ctDNA) assessment predict outcome and resistance in hormone-receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients (pts) treated with CDK4/6 inhibitor (CDK4/6i) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-01.

Association between low fetal fraction of cell free DNA and adverse pregnancy outcomes

Association between low fetal fraction of cell free DNA and adverse pregnancy outcomes

Clinical application of non-invasive prenatal diagnosis of phenylketonuria based on haplotypes via paired-end molecular tags and weighting algorithm

BackgroundPhenylketonuria (PKU) is a metabolic disease that can cause severe and irreversible brain damage without treatment.MethodsHere we developed a non-invasive prenatal diagnosis (NIPD) technique based on haplotypes via paired-end molecular tags and weighting algorithm and applied it to the NIPD of PKU to evaluate its accuracy and feasibility in the early pregnancy. A custom-designed hybridization probes containing regions in phenylalanine hydroxylase (PAH) gene and its 1 Mb flanking region were used for target sequencing on genomic and maternal plasma DNA (7–13 weeks of gestation) to construct the parental haplotypes and the proband’s haplotype. Fetal haplotype was then inferred combined with the parental haplotypes and the proband’s haplotype. The presence of haplotypes linked to both the maternal and paternal mutant alleles indicated affected fetuses. The fetal genotypes were further validated by invasive prenatal diagnosis in a blinded fashion.ResultsThis technique has been successfully applied in twenty-one cases. Six fetuses were diagnosed as patients carrying both of the mutated haplotypes inherited from their parents. Eleven fetuses were carriers of one heterozygous PAH variants, six of which were paternal and five of which were maternal. Four fetuses were absence of pathogenic alleles. All results were consistent with the prenatal diagnosis through amniotic fluid.ConclusionsThe results showed that our new technique applied to the genotyping of fetuses with high risk for PKU achieves an accurate detection at an early stage of pregnancy with low fetal fraction in cell free DNA.

Screening for Fetal Aneuploidy and Sex Chromosomal Anomalies in a Pregnant Woman With Mosaicism for Turner Syndrome-Applications and Advantages of Cell-Based NIPT.

Background: Cell-free NIPT and cell-based NIPT are risk-free testing options using maternal blood samples to screen for fetal aneuploidies, but the methods differ. For cell-free NIPT, the fetal fraction of cell-free DNA in plasma is analyzed with a high background of maternal DNA. In contrast, for cell-based NIPT, a limited number of the rare, intact fetal cells are isolated for the genetic analysis. This case demonstrates the differences regarding testing for fetal sex-chromosomes anomalies (SCAs) between these two tests.Materials and Methods: A pregnant woman with mosaicism for Turner syndrome opted for NIPT in first trimester. For the cell-free NIPT analysis, DNA extraction, genome-wide massive parallel sequencing, and data analysis were carried out as described by the kit manufacturer (Illumina©, San Diego, CA, USA). For cell-based NIPT, the first sample gave no result, but the woman consented to repeat cell-based NIPT. After whole genome amplification and STR analysis, fetal DNA from three individual fetal cells was subjected to chromosomal microarray (aCGH, Agilent oligoarray, 180 kb).Results: Fetal fraction was 7%, and cell-free NIPT showed 2 copies of chromosomes 13, 18, and 21 and a decreased proportion of chromosome X, suggestive of fetal Turner syndrome. In contrast, the cell-based NIPT result showed no aneuploidy and two X-chromosomes in the fetus.Conclusion: cell-based NIPT may provide a non-invasive testing option to screen for SCAs in women with mosaicism for monosomy-X in blood, where cell-free NIPT cannot discriminate whether the X-loss is maternal or fetal.