cfDNA Results Research Articles

Clinical Examples of the Additive Value of Absolute Quantification of Cell-Free DNA After Heart Transplantation.

Cell-free DNA (cfDNA) is used as a biomarker after transplantation to detect graft injury, relying on the donor fraction (DF). We have established a PCR-based approach allowing us to separately quantify absolute values of dd-cfDNA and recipient-derived cfDNA (rd-cfDNA). We aimed to present typical clinical scenarios after heart transplantation (HTx) to illustrate the advantages of absolute cfDNA values over DF. We used the cfDNA results of our cohort (509 samples of 52 patients followed during the first year after HTx) as background and determined the trajectories of cfDNA in specific clinical situations. We profiled an uncomplicated clinical course, viral and bacterial infections, acute and chronic rejection, and false-negative and false-positive rejections in six patients (five adults, one child). There was a substantial discrepancy between relative (DF) and absolute cfDNA-levels in several clinical situations. Rd- and dd-cfDNA were independently elevated during episodes of rejection and infection and were better suited to depict treatment response than DF alone. Absolute quantification of cfDNA may offer clinically relevant information additive to DF in various situations after HTx and could be helpful for more accurate monitoring of diagnosis and treatment of rejection.

Discordant Prenatal Cell-Free DNA Screening vs. Diagnostic Results of Sex Chromosome Aneuploidies: Implications for Newborn Screening and Genetic Counseling.

Sex chromosome aneuploidies (SCAs) collectively occur in 1 in 500 livebirths, and diagnoses in the neonatal period are increasing with advancements in prenatal and early genetic testing. Inevitably, SCA will be identified on either routine prenatal or newborn screening in the near future. Tetrasomy SCAs are rare, manifesting more significant phenotypes compared to trisomies. Prenatal cell-free DNA (cfDNA) screening has been demonstrated to have relatively poor positive predictive values (PPV) in SCAs, directing genetic counseling discussions towards false-positive likelihood rather than thoroughly addressing all possible outcomes and phenotypes, respectively. The eXtraordinarY Babies study is a natural history study of children prenatally identified with SCAs, and it developed a longitudinal data resource and common data elements with the Newborn Screening Translational Research Network (NBSTRN). A review of cfDNA and diagnostic reports from participants identified a higher than anticipated rate of discordance. The aims of this project are to (1) compare our findings to outcomes from a regional clinical cytogenetic laboratory and (2) describe discordant outcomes from both samples. Twenty-one (10%), and seven (8.3%) cases were found to be discordant between cfDNA (result or indication reported to lab) and diagnosis for the Babies Study and regional laboratory, respectively. Discordant results represented six distinct discordance categories when comparing cfDNA to diagnostic results, with the largest groups being Trisomy cfDNA vs. Tetrasomy diagnosis (66.7% of discordance in eXtraordinarY Babies study) and Mosaicism (57.1% in regional laboratory). Traditional genetic counseling for SCA-related cfDNA results is inadequate given a high degree of discordance that jeopardizes the accuracy of the information discussed and informed decision making following prenatal genetic counseling.

P-543 Multicenter concordance study of embryo cell-free DNA and trophectoderm biopsies: impact on clinical outcomes

Abstract Study question Are reproductive outcomes of transfers from euploid day-6 biopsied blastocysts different depending on results of the corresponding embryo cell-free DNA (cfDNA) in the culture medium? Summary answer Transfers with euploid trophectoderm biopsies but aneuploid cfDNA showed higher miscarriage rates than those with euploid trophectoderm biopsies and cfDNA, although without reaching statistical significance. What is known already During development, embryos release cfDNA. There is, therefore, genetic material in the droplet of culture medium in which they develop. When analyzing, for the same embryo, the spent blastocyst medium containing cfDNA, trophectoderm (TE) and inner cell mass biopsies or the whole blastocyst, high concordance rates have been observed. This high representativeness shown by cfDNA on the chromosomal content of the blastocysts opens a new era of possibilities for non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) as its analysis avoids the need of an invasive trophectoderm biopsy for aneuploidy detection. Study design, size, duration Prospective, observational, multicenter study conducted in 10 assisted reproductive centers after approval by each local institutional review board. From April 2018 to December 2022, 2539 embryos from 716 patients included in PGT-A cycles were cultured following a niPGT-A culture protocol. Blastocysts underwent TE biopsy and media collection that were subsequently analyzed and compared. 441 frozen single embryo transfers (SET) from euploid embryos, based on the TE biopsy result, were performed and their clinical data registered. Participants/materials, setting, methods Embryos were cultured in routine conditions up to day 4, then washed and transferred to a new 10μl droplet. On day 6, media were collected and frozen at -20 °C; and blastocyst biopsy and vitrification were performed. Samples were analyzed by NGS (Ion ReproSeq PGS kit, ThermoFisher Scientific) and the results processed with customized algorithms for TE/cfDNA. SET were performed based on TE biopsy results, with blinded cfDNA results at the moment of transfer. Main results and the role of chance Clinical outcomes were compared according to whether the TE and cfDNA result was euploid (eu-eu) or with euploid TE and aneuploid cfDNA (eu-aneu). Transfers where cfDNA analysis had provided non-informative results were excluded. The eu-eu group included 288 transfers (mean female age = 35.3 ± 4.9 years), with an ongoing pregnancy rate (OPR) of 44.4% and a miscarriage rate (MR) of 14.7%. Whereas the eu-aneu group included 95 transfers (mean female age = 34.4 ± 5.2 years), with an OPR of 43.2% and a MR of 25.5%. The comparisons of OPR and MR between both groups did not reach statistical significance (Chi-square test), but MR showed a two-fold increase in transfers with aneuploid cfDNA. A sub-analysis was performed, while only considering transfers from patients without endometrial factor history. Eu-eu and eu-aneu groups included 231 and 73 transfers, respectively. The differences in OPR (49.4% vs 43.8%) and MR (14.3% vs 25.6%) were slightly higher between both groups but did not reach statistical significance (Chi-square test). Limitations, reasons for caution To better understand the trends observed, especially on the miscarriage rate, it would be necessary to collect clinical outcomes from more SET. This study could not determine the benefit of embryo selection based only in cfDNA, since SET was performed according to TE biopsy results. Wider implications of the findings The analysis of embryo cfDNA could provide useful information to help select the best embryo for transfer and, therefore, decrease miscarriage rates and increase the chances of having a healthy newborn at home. Trial registration number NCT03520933

Cell-free DNA test for fetal chromosomal abnormalities in multiple pregnancies.

This study aimed to report the screening performance of cell-free DNA (cfDNA) testing for chromosomal abnormalities in twins, triplets, and vanishing twin pregnancies. Data were obtained from pregnant women with a multiple pregnancy or a vanishing twin pregnancy at ≥10 weeks' gestation who requested self-financed cfDNA testing between May 2015 and December 2021. Those that had positive screening results had diagnostic confirmatory procedures after counseling and consent. The performance of screening of the cfDNA test was determined by calculating confirmation rate and combined false-positive rate (cFPR). Data from 292 women were included after exclusion of those lost to follow-up, with no-result on cfDNA testing, or had reductions. Of the 292 pregnancies, 10 (3.4%) were triplets, including no cases of trisomy 21 and trisomy 18; 249 (85.3%) were twins, including 3 cases of trisomy 21 and no cases of trisomy 18 and 13; and 33 (11.3%) were vanishing twins, including 3 cases of trisomy 21 and 1 case of trisomy 18. The median (IQR) maternal age was 34 years (31-37). For triplet pregnancies, the initial no-result rate was 10.3% (95% confidence interval [CI] 3.6-26.4), all with results after redraw. For twin pregnancies, the initial no-result rate was 12.9% (95% CI 9.6-17.0), and the no-result rate after redraw was 1.6% (95% CI 0.7-3.6). For vanishing twins, there were no cases with no-result. All triplets had low-risk cfDNA results. The confirmation rate for trisomy 21 was 100% with a FPR at 0% due to the small number of positive cases for twins. For vanishing twins, one high-risk case for trisomy 21 and the only high-risk case for trisomy 18 were confirmed with a cFPR of 8.3% (n = 2/24; 95% CI 2.3-25.9). cfDNA testing in twin pregnancies has sufficient screening performance for trisomy 21 but the number of affected cases for other conditions is limited to draw any meaningful conclusion. The use of cfDNA testing in triplet pregnancies and vanishing twins remains an area for further research.

Diagnostic accuracy of cell-free DNA in maternal blood in detecting chromosomal anomalies in twin pregnancies: systematic review and meta-analysis.

To report the diagnostic accuracy of cell-free DNA (cfDNA) in maternal blood in detecting chromosomal anomalies in twin pregnancies. Medline, Embase and Cochrane databases were searched. The inclusion criteria were twin pregnancies undergoing cfDNA screening for Trisomies 13, 18, 21, monosomy X0 and other sex chromosomal anomalies (SCA). The index test was represented by a positive results of cfDNA test. The reference standard was represented by the karyotype results (obtained either pre or postnatally) or, in case of negative cfDNA result, by a normal neonatal phenotype. The quality of the studies was assessed using the revised tool for the quality assessment of diagnostic accuracy studies (QUADAS-2). Summary estimates of sensitivity, specificity, positive and negative likelihood ratios (LR+ and LR-) and diagnostic odds ratio (DOR), with the corresponding 95% Confidence Intervals (95% CI), were computed using the bivariate random-effects model. Thirty-five studies were included. cfDNA had an overall high accuracy in detecting Trisomy 21 in twin pregnancies with a sensitivity of 98.8% (95% CI 96.5-100), a specificity of 100% (95% CI 99.9-100). Sensitivity and specificity were of 94.9% (95% CI 75.6-99.1) and 100 (95% CI 99.9-100) for Trisomy 18, and 84.6% (95% C% 54.6-98.1) and 100% (95% CI 99.9-100) for Trisomy 13 . We could not compute the diagnostic accuracy of cfDNA in detecting monosomy X0 in twins, while cfDNA had a sensitivity of 100% (95% CI 71.5-100) and a specificity of 99.8% (95% CI 99.7-99.9) in detecting other SCA (11 cases). The accuracy of cfDNA in detecting Trisomy 21, 18 and 13 was similar in dichorionic and monochorionic twin pregnancies. cfDNA has a high diagnostic accuracy in detecting Trisomy 18 and 21 in twin pregnancies, irrespective of chorionicity. Accuracy in the detection of Trisomy 13 and SCA was limited by the small number of affected cases and the difficulties in the confirmation of false negative cases in case of SCA and requires confirmation in larger studies. This article is protected by copyright. All rights reserved.

Detection of tumor-derived cell-free DNA in cerebrospinal fluid using a clinically validated targeted sequencing panel for pediatric brain tumors.

Clinical sequencing of tumor DNA is necessary to render an integrated diagnosis and select therapy for children with primary central nervous system (CNS) tumors, but neurosurgical biopsy is not without risk. In this study, we describe cell-free DNA (cfDNA) in blood and cerebrospinal fluid (CSF) as sources for "liquid biopsy" in pediatric brain tumors. CSF samples were collected by lumbar puncture, ventriculostomy, or surgery from pediatric patients with CNS tumors. Following extraction, CSF-derived cfDNA was sequenced using UW-OncoPlex™, a clinically validated next-generation sequencing platform. CSF-derived cfDNA results and paired plasma and tumor samples concordance was also evaluated. Seventeen CSF samples were obtained from 15 pediatric patients with primary CNS tumors. Tumor types included medulloblastoma (n = 7), atypical teratoid/rhabdoid tumor (n = 2), diffuse midline glioma with H3 K27 alteration (n = 4), pilocytic astrocytoma (n = 1), and pleomorphic xanthoastrocytoma (n = 1). CSF-derived cfDNA was detected in 9/17 (53%) of samples, and sufficient for sequencing in 8/10 (80%) of extracted samples. All somatic mutations and copy-number variants were also detected in matched tumor tissue, and tumor-derived cfDNA was absent in plasma samples and controls. Tumor-derived cfDNA alterations were detected in the absence of cytological evidence of malignant cells in as little as 200µl of CSF. Several clinically relevant alterations, including a KIAA1549::BRAF fusion were detected. Clinically relevant genomic alterations are detectable using CSF-derived cfDNA across a range of pediatric brain tumors. Next-generation sequencing platforms are capable of producing a high yield of DNA alterations with 100% concordance rate with tissue analysis.

Cell Free Microbial DNA Utilization at a Children’s Hospital

Background: We investigated the utilization of cell free microbial DNA (cfDNA) at a Children’s Hospital. Materials and Methods: cfDNA results were assessed regarding the contribution to therapeutic decisions. Results: Of 80 tests on 59 children, 1 test was unevaluable. At least one agent was identified in 45/79 (57%) tests from 34/59 (58.2%) children, 34/79 (43.0%) were negative in 31/59(52.5%) children. Of 45 positive results, 24/79 (30%) were contributory, 15/79 (19%) were diagnostic, 6/79 (7.6%) were diagnostic but diagnosis could have been made with other testing modalities, and 3/79 (3.8%) were diagnostic with minimal previous workup. 21/79 (26.6%) positives were noncontributory. Of 35 negative results, 9/79 (11.4%) were contributory, 26/79 (33.0%) were noncontributory. Efficiency was 30.4–41.8%. cfDNA detected agents not detected by conventional techniques in 22/79 (27.8%), detected different agents in 9/79 (11.4%), and failed to detect agents identified by conventional techniques in 4 (5%). Conclusions: Efficiency of cfDNA was 30.4–41.8%

Circulating tumor DNA (ctDNA) positivity and its association with clinicopathological characteristics by novel blood-based test for colorectal cancer (CRC) screening from a multi-center large cohort: COSMOS-CRC-01.

69 Background: Blood-based CRC screening has yielded performance comparable to stool-based screening tests and may be a modality that addresses the adherence gap in CRC screening. To investigate the diagnostic performance and its clinical relevance, we evaluated the test performance of a blood-based CRC screening test in a large cohort of individuals enriched for advanced colorectal neoplasia and early CRCs. Methods: 501 individuals presenting to ten institutions in Japan from 2020 to 2021 for treatment of advanced adenomas (AAs) or CRCs were consented and provided a pre-treatment blood sample (40mL whole blood in Streck cfDNA blood collection tubes). All eligible individuals underwent surgery or endoscopic resection within 30 days after sample collection and the pathological specimens were reviewed to determine cancer histology and stage, as well as AA histology. Pre-treatment blood samples were analyzed with a multi-modal (genomic and epigenomic) cfDNA CRC detection assay (Shield, Guardant Health, USA). Final cfDNA results were correlated with clinical data to determine the association of clinicopathological characteristics with test sensitivity. This study is registered as a clinical trial under UMIN000037765. Results: 451/501 (90%) individuals met inclusion/exclusion criteria and provided adequate blood samples that passed testing quality control criteria. Median age at consent was 67.4 years (range 31 – 92). 44% were female. 214/451 (47%) had a prior positive fecal occult blood test (FOBT). Of individuals with a negative or unknown FOBT testing, 161/237 (68%) had GI symptoms (e.g. abdominal pain, bloody/narrowing stool, constipation/diarrhea). The test identified 88% (268/305) of CRCs, including 71% (79/111) Stage I, 98% (87/89) Stage II, 97% (90/93) Stage III, and 100% (12/12) Stage IV. Overall sensitivity in detecting Stage I-III was 87%. The test identified 37% (54/146) of AAs, including 49% (33/68) of carcinoma in situs and 24% (9/37) of AAs with high-grade dysplasia. Sensitivity for right-sided CRC was 84% (61/73), 87% (59/68) for left-sided CRC, and 90% (148/164) for rectal tumors. Stage I test sensitivity was significantly associated with tumor size (>20mm; p<0.01), left-sided or rectal tumor location (p<0.05), and tumor depth (p<0.01) in univariate analysis, whereas the tumor depth (T2) was the sole factor associated with test positivity (p<0.05) in multivariate analysis. Specificity on this assay is 90% as determined in the ECLIPSE clinical study (NCT04136002). Conclusions: This study evaluates the performance of a blood-based test for CRC and its clinicopathological relevance in a large-scale cohort. In this population enriched for early colorectal neoplasia, the sensitivity was 88% for CRC and 37% for AAs. This performance is consistent with previous reports. Clinical trial information: UMIN000037765 .

The Predictive Value of Prenatal Cell-Free DNA Testing for Rare Autosomal Trisomies: A Systematic Review and Meta-analysis

ABSTRACT Noninvasive prenatal testing has been a reliable method for prenatal screening for the common autosomal trisomies for the last decade. It has been analyzed in many prospective studies for accuracy in screening for the most common trisomies—13, 18, and 21—and the technology has continued to progress. With high accuracy for common trisomies, advances are attempting to expand noninvasive prenatal testing for identifying rare autosomal trisomies, such as trisomies 7, 15, 16, and 22; sex chromosome aneuploidies; and segmental copy number variants. Although cell-free DNA (cfDNA) technology is now available to screen for these abnormalities, accuracy and detection rates have not been well studied. This systematic review analyzes and reports the diagnostic accuracy of cfDNA screening for the rare autosomal trisomies. This review included case series with 10 or more cases that reported on the accuracy of cfDNA in reporting rare autosomal trisomies that were confirmed by diagnostic results or postnatal genetic testing; the final review included 31 studies. Methodological analysis found the risk of bias to be high in most studies, with only 5 rated as high quality and low risk in all domains. All studies included in the final analyses were published between 2017 and 2022, and study populations ranged from 14 to 153,575 patients. Indications for screening and characteristics of the patients varied from study to study, with 12 studies in high-risk women, 10 studies in the general obstetric population, and 9 studies without inclusion of underlying risk factors and indications. Whole-genome sequencing was used in all studies as testing methodology. Testing timing varied and was performed anywhere from gestational week 8 to week 39. In the 3 studies that analyzed sensitivity and specificity of cfDNA testing, sensitivity ranged from 87.2% to 100%, and specificity ranged from 90.7% to 99.9%. In all 3 of these studies, there were significant limitations that led authors to acknowledge the difficulty of drawing clear conclusions about sensitivity and specificity from their data. Meta-analysis performed for positive predictive value (PPV) provided a pooled estimate of 11.46% (95% confidence interval [CI], 7.80%–15.65%). Sensitivity analysis including only studies at low risk of bias gave a PPV of 9.13% (95% CI, 2.49%–18.76%). Analysis was performed again with groups divided into high or low background risk, with a slightly higher PPV in the higher background risk group, but no significant difference between these groups (P = 0.595). Statistical heterogeneity was not explained by year of publication or baseline risk of participants but could be largely explained by rate of diagnostic follow-up. Studies with a follow-up rate of less than 50% showed a PPV of 23.69% (95% CI, 9.21%–41.94%), whereas studies with a follow-up rate greater than 50% reported a PPV of 8.80% (95% CI, 6.08%–11.90%). Based on the results of this systematic review and meta-analysis, it is likely that previously reported PPVs of cfDNA's diagnostic accuracy for detecting rare autosomal trisomies are overestimated. A reason for this could be lower follow-up rates as well as testing bias in women with high-risk cfDNA results. One reason for the low prevalence of rare autosomal trisomies is that many confirmed cases result in miscarriage before 12 weeks of gestation. Pregnancies that continue and that are not mosaic often experience major complications. The occurrence of mosaicism may impact the PPV, as mosaicism carries some risks but is sometimes not detected from cfDNA samples. This study was limited by the details included in the studies analyzed; this caused significant statistical heterogeneity, which limits the conclusions that can be drawn. In addition, detection rates may differ by type of cfDNA test, and this analysis did not stratify by type of test. The results of this analysis are consistent with previous literature on detection of rare autosomal trisomies in the general population and show that even with a high specificity and sensitivity for cfDNA testing in general, care should be taken in making clinical decisions on the part of both providers and patients in instances where a rare autosomal trisomy is suspected.

Analysis of retest reliability for pregnant women undergoing cfDNA testing with a no-call result

BackgroundDetermining the reasons for unreportable or no-call cell-free DNA (cfDNA) test results has been an ongoing issue, and a consensus on subsequent management is still lacking. This study aimed to explore potential factors related to no-call cfDNA test results and to discuss whether retest results are reliable.Methods and resultsThis was a retrospective study of women with singleton pregnancies undergoing cfDNA testing in 2021. Of the 9871 pregnant patients undergoing cfDNA testing, 111 had a no-call result, and their results were compared to those of 170 control patients. The no-call rate was 1.12% (111/9871), and the primary cause for no-call results was data fluctuation (88.29%, 98/111). Medical conditions were significantly more frequent in the no-call group than in the reportable results group (P < 0.001). After retesting, 107 (107/111, 96.40%) patients had a result, and the false-positive rate (FPR) of retesting was 10.09% (10.09%, 11/109). In addition, placental lesions were more frequent in the no-call group than in the reportable results group (P = 0.037), and 4 patients, all in the no-call group, experienced pregnancy loss.ConclusionsPregnant women with medical conditions are more likely to have a no-call result. A retest is suggested for patients with a no-call result, but retests have a high FPR. In addition, pregnant women with a no-call result are at increased risk of adverse pregnancy outcomes. In conclusion, more attention should be given to pregnant women for whom a no-call cfDNA result is obtained.

Incidence of sex chromosome aneuploidy in a prenatal population: 27-year longitudinal study in Northern Italy.

The availability of cell-free (cf) DNA as a prenatal screening tool affords an opportunity for non-invasive identification of sex chromosome aneuploidy (SCA). The aims of this longitudinal study were to investigate the evolution and frequency of both invasive prenatal diagnostic testing, using amniocentesis and chorionic villus sampling (CVS), and the detection of SCA in cfDNA samples from a large unselected cohort in Northern Italy. The results of genetic testing from CVS and amniotic fluid samples received from public and private centers in Italy from 1995 to 2021 were collected. Chromosomal analysis was performed by routine Q-banding karyotype. Regression analyses and descriptive statistics were used to determine population data trends regarding the frequency of prenatal diagnostic testing and the identification of SCA, and these were compared with the changes in indication for prenatal diagnostic tests and available screening options. Over a period of 27 years, there were 13 939 526 recorded births and 231 227 invasive procedures were performed, resulting in the prenatal diagnosis of 933 SCAs. After the commercial introduction of cfDNA use in 2015, the frequency of invasive procedures decreased significantly (P = 0.03), while the frequency of prenatal SCA detection increased significantly (P = 0.007). Between 2016 and 2021, a high-risk cfDNA result was the indication for 31.4% of detected sex chromosome trisomies, second only to advanced maternal age. Our findings suggest that the inclusion of SCA in prenatal cfDNA screening tests can increase the prenatal diagnosis of affected individuals. As the benefits of early ascertainment are increasingly recognized, it is essential that healthcare providers are equipped with comprehensive and evidence-based information regarding the associated phenotypic differences and the availability of targeted effective interventions to improve neurodevelopmental and health outcomes for affected individuals. © 2023 International Society of Ultrasound in Obstetrics and Gynecology.

P-737 Progress in non-invasive embryo assessment with cell-free nucleic acids in blastocoel fluid

Abstract Study question Can cell-free nucleic acids (cfDNA) from blastocoel fluid (BF) after a trophectoderm (TE) biopsy be used as a non-invasive sample type to assess embryos? Summary answer Both BF and spent media carry comparable, highly fragmented cfDNA and cell-free RNA (cfRNA) which may reveal more about the quality of the embryo. What is known already Preimplantation genetic testing for aneuploidy (PGT-A) is commonly used by IVF clinics to screen embryos for any chromosomal abnormalities during the IVF process. Noninvasive preimplantation genetic testing (ni-PGT) is an emerging category of tools within the field of assisted reproductive technology. Currently, niPGT products have been developed for embryo aneuploidy detection using cfDNA released by the embryo into the spent media. Embryo spent media (ESM) carries cfDNA, which has the potential to be applied to evaluate the quality of the embryo. Previous studies have demonstrated that cfDNA in ESM is highly fragmented to the nucleosome level. Study design, size, duration The quality and quantity of cell free nucleic acid from forty BF-conditioned media (BFCM) samples were analyzed in this study. Embgenix™ Analysis Software was used to analyze and interpret sequencing data. For each sample, the concentration and fragmentation of cfDNA were examined and compared using generalized linear mixed models. To assess RNA quality at a picogram level, reverse-transcribed and amplified cDNA was used to represent the quality of cfRNA in BFCM. Participants/materials, setting, methods Embryos were washed and placed in individual biopsy media droplets for TE biopsy. After biopsy, blastocysts were collapsed, releasing BF into the droplet. The entire droplet was collected as BFCM. The Embgenix ESM Screen Kit was used to prepare sequencing libraries. The SMART-Seq® Single Cell Kit was used to evaluate the RNA quality. The BFCM cfDNA results were also compared to spent media profile that had already been obtained from a different cohort of embryos. Main results and the role of chance Amounts of cfDNA fragmentation detected in BFCM samples was comparable to ESM. Whole-genome amplification (WGA) yields correlated to the amount of cfDNA input but not the degree of cfDNA fragmentation. Informative sequencing reads and computed copy number variations (CNV) plot noise, as evaluated by distribution of log2 ratio spread (DLRS), also showed correlation and stable performance down to ∼2 pg of cfDNA input. We extracted detectable cfRNA from BFCM and created cDNA for RNA quality analysis. Our model demonstrates a significant correlation between cDNA length and RNA quality metrics, with a low DV200 of cfRNA from BFCM samples. The quantity and quality of cfDNA released into spent culture media by a developing embryo are comparable to the cfDNA released into BFCM upon TE biopsy, indicating that BFCM has potential as a noninvasive method for assessing the ploidy status of embryos. WGA yield, informative reads, and DLRS of BFCM samples were likewise comparable to data previously acquired using spent media samples. However, the severely fragmented cfRNA in these samples illustrate the inherent instability of RNA and the difficulty of utilizing RNA analysis to determine embryo health. Assays compatible with small RNA or microRNA (miRNA) are more appropriate for RNA-seq of BFCM. Limitations, reasons for caution Different clinics use varying methods for embryo culture and biopsy; the data reported here is from a single research center. The characteristic of samples may vary between clinics. Our assay estimates RNA quality using amplified cDNA; nonetheless, discrepancies between fragmented RNA and amplified cDNA may exist. Wider implications of the findings In PGT-A, BFCM samples are routinely discarded. Our findings imply BFCM samples contain considerable levels of cell-free nucleic acids which could also be used to screen embryo quality. BFCM samples, therefore, could be analyzed in conjunction with PGT-A to select and/or rank embryos for implantation. Trial registration number Not applicable

NeoRAS wild-type metastatic colorectal cancer in the SCRUM-Japan GOZILA study.

3506 Background: The ’’Neo RAS’’ phenomenon refers to RAS mutant (MT) metastatic colorectal cancer (mCRC) that becomes RAS wild-type (WT) following treatment. This Neo RAS WT population might represent a novel indication for EGFR inhibitors, which are less effective in RAS MT mCRC. The incidence and clinicopathological characteristics of Neo RAS WT mCRC using plasma cell-free DNA (cfDNA) next generation sequencing has not been defined. Methods: As part of a large-scale nationwide screening platform (SCRUM-Japan GOZILA), 478 patients with an initial diagnosis of RAS MT mCRC by tissue analysis (MEBGEN RASKET-B) who received systemic therapy underwent cfDNA testing (Guardant 360) prior to later lines of treatment. Based on the cfDNA results, we evaluated the clinicopathological characteristics of those with Neo RAS WT and RAS MT. Neo RAS WT was defined as no RAS MT ( KRAS or NRAS) detected in plasma and was assessed in the overall cohort (Cohort A) and in the subgroup with at least one somatic alteration detected in plasma (Cohort B) to exclude those with insufficient tumor DNA shedding. Results: Median age at the time of blood sampling was 62.0 years old, and 257 (51.9%) were men. 160 (32.3%) and 319 (64.4%) had right-sided tumors and multi-organ metastases. The lungs were the most frequent site of metastasis (60.2%), followed by liver (57.4%), lymph nodes (28.9%), and peritoneum (28.5%). The prevalence of Neo RAS WT was 19.0% (91/478) in Cohort A and 9.8% (41/429) in Cohort B. The frequency of Neo RAS WT in tumors originally with KRAS exon 2 MT tended to be lower than in those with other RAS MT (18.1% vs 25.4%, P = 0.21 in Cohort A, 9.0% vs 15.4%, P = 0.14 in Cohort B). There were significant differences in the prevalence of Neo RAS WT between patients with single organ vs multi-organ metastases (P &lt; 0.001 in Cohort A, P = 0.004 in Cohort B), absence vs presence of liver metastasis (P &lt; 0.001 in both Cohort A and B), lymph node metastasis (P = 0.006 in Cohort A), peritoneal metastasis (P = 0.002 in group A), and bone metastasis (P = 0.029 in Cohort A), testing immediately prior to 2nd through to 4th line treatment vs later lines (P = 0.007 in Cohort A), immediate prior use of regorafenib (P = 0.027 in Cohort A) and any history of vascular endothelial growth factor inhibitors (P = 0.003 in Cohort A, P = 0.035 in Cohort B). In the logistic regression multivariate analysis, absence of liver metastasis (odds ratio [OR], 5.83; P &lt; 0.001 in Cohort A, OR, 2.84; P = 0.005 in Cohort B), absence of lymph node metastasis (OR, 2.18; P = 0.034 in Cohort A) and tissue RAS MT other than KRAS exon 2 (OR, 2.35; P = 0.049 in Cohort B) were significantly related to the emergence of Neo RAS WT. Among 6 Neo RAS WT patients tretd with EGFR inhibitors, one had partial response and another one had stable disease for at least 6 months. Conclusions: Liver and lymph node metastasis and RAS MT other than those in KRAS exon 2 are factors associated with the development of Neo RAS WT mCRC. EGFR inhibitors might be effective treatment.

Abstract P4-01-14: Changes in the Genomic Spectrum of Actionable Alterations in HER2 Negative Metastatic Breast Cancer in Serial Cell Free DNA (cfDNA) Analysis

Abstract Background: Serial cell free DNA (cfDNA) analysis in metastatic breast cancer (MBC) is a noninvasive method for tracking tumor evolution and the emergence of new somatic alterations through the course of the disease. The detection of new actionable alterations may impact treatment selection; thus, serial cfDNA collection is increasingly being performed in the clinic. However, it is not well-understood how often serial cfDNA testing identifies new actionable alterations. We evaluated changes in the genomic spectrum of actionable alterations in serial cfDNA analysis of HER2 negative (HER2-) MBC. Method: Patients with HER2- MBC who underwent plasma-based cfDNA testing (Guardant360, 74-gene assay) between February 2015 and February 2021 at an academic institution were included. A retrospective review of records was conducted to identify subtype, demographics, lines of therapy, and cfDNA results. At baseline cfDNA testing, all cfDNA alterations were considered new. For patients with serial draws, new cfDNA alterations in each draw, compared to previous draws, were quantified and characterized. The pathogenicity of new alterations was determined using the OncoKB precision oncology database. New pathogenic alterations were further classified as actionable alterations (AA) using the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). Alterations that met the ESCAT I (alteration-drug match was associated with improved outcomes in clinical trials) or ESCAT II (alteration-drug match was associated with antitumor activity) criteria were considered as AA. Results: 344 patients with hormone receptor positive (HR+) MBC and 95 patients with triple negative (TN) MBC had a baseline cfDNA draw. Among the HR+ cohort, 139, 79 and 48 patients had 2nd, 3rd, and 4th serial cfDNA draws, respectively. Among the TN cohort, 33 and 18 patients had 2nd and 3rd serial cfDNA draws, respectively. Table 1 depicts the prevalence of AA found in each of these draws, as well as the median number of prior therapies at each point of collection and the mean time between serial draws. The median number of new genomic alterations was lower in subsequent draws compared to the baseline, regardless of subtype (HR+: 4 vs. 2, TN: 4 vs. 1.5-3, at baseline vs. subsequent draws, respectively). In the HR+ cohort the proportion of patients with new AA (ESCAT I/II) decreased from 63% at baseline to 27-33% in the 2nd-4th draws. While some of the new AA in subsequent draws were new actionable variants in the same genes that were known to be altered in previous draws (in 19%, 68% and 57% of patients with new AA in the 2nd, 3rd and 4th draws, respectively), the remainder were new AA in previously unaltered genes. In the TN cohort 25% of patients had new AA at baseline and this proportion of TN patients with new AA continued to decrease in subsequent draws (9% and 0% in the 2nd and 3rd draws, respectively). PIK3CA and ESR1 were the most frequent genes with new AA in the ER+ cohort, regardless of draw number. In the TN cohort, PIK3CA was the most frequent gene with new AA in the first draw, and in the second draw, new AA in PIK3CA, BRCA2, and ERBB2 were present at an equal frequency. Conclusion: While the proportion of patients with HER2- MBC identified to have new AA in serial cfDNA decreased with time for both HR+ and TNBC, new alterations continue to emerge, particularly for patients with HR+ MBC. Further research is needed to determine the impact of serial cfDNA testing on the selection of genotype-matched therapy and outcomes. Table 1. Citation Format: Yael Bar, Jennifer C. Keenan, Lianne Ryan, Dejan Juric, Jennifer Shin, Seth A. Wander, Laura M. Spring, Beverly Moy, Leif Ellisen, Steven J. Isakoff, Aditya Bardia, Neelima Vidula. Changes in the Genomic Spectrum of Actionable Alterations in HER2 Negative Metastatic Breast Cancer in Serial Cell Free DNA (cfDNA) Analysis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-14.

Abstract PD1-10: Impact of Race on Clinical, Socioeconomic, and Genomic Characteristics, Clinical Trial Participation, and Receipt of Genotype-matched Therapy Among Patients with Metastatic Breast Cancer

Abstract Background: Clinical outcomes in breast cancer differ across racial and ethnic populations. We have previously demonstrated that receipt of genotype-matched therapy targeted to an actionable mutation may potentially improve patient outcomes (Vidula, CCR, 2021). We evaluated the impact of race on clinical, socioeconomic, and genomic characteristics, clinical trial participation, and receipt of genotype-matched therapy among patients with metastatic breast cancer (MBC). Methods: We conducted a retrospective study of patients with MBC at an academic institution who underwent cell-free DNA testing (cfDNA, Guardant360, 74 gene panel) as part of routine clinical care from 11/29/2016-11/2/2020. Patient demographics (including self-reported race and ethnicity) and clinical trial enrollment (at same institution) were determined by retrospective data collection. Mutations identified in cfDNA were characterized as actionable based on the variant interpretation performed by Guardant360 using vetted genomic databases, and receipt of genotype-matched therapy targeted to an actionable mutation was determined as previously described (Vidula, CCR, 2021). Pearson’s chi-squared and Wilcoxon rank-sum tests were used to compare categorical and continuous variables between groups, with p&amp;lt; 0.05 indicating statistical significance. Results: Four hundred and twenty-five patients with MBC and cfDNA results were identified, of which 369 were White (87%), 27 Black (6.4%), 15 Hispanic (3.5%), and 14 Asian (3.3%). There were no significant differences in median age at MBC diagnosis (p=0.064), disease subtype distribution (p=0.74), proportions of de-novo/recurrent MBC (p=0.95), presence of visceral metastases (p=0.84), Charleston comorbidity index (p=0.93), menopausal status (p=0.3), and level of education (p=0.44) across racial groups. Higher proportions of non-primary English speakers were seen in Hispanic (80%) and Asian (29%) races (p&amp;lt; 0.001). Median distance traveled to the institution also varied based on race, with White patients traveling further (White: 39.1 miles, Black: 21.8 miles, Hispanic 9.4 miles, Asian 9.1 miles, p&amp;lt; 0.001). In addition, type of insurance varied based on race, with White patients having the highest rates of commercial insurance and Medicare, Black patients having the highest rate of state-supported insurance, and Asian patients having the highest uninsured rates (p&amp;lt; 0.001). Clinical trial enrollment rates did not significantly differ by race (White: 44%, Black: 37%, Hispanic: 47%, and Asian 21%, p=0.34), but patients without insurance were significantly less likely to be enrolled on a trial than those with commercial insurance (p=0.03). The proportion of patients with ≥1 actionable mutation in cfDNA did not vary significantly by race (White: 78%, Black: 56%, Hispanic: 73%, Asian 86%, p=0.18) and the median number of actionable mutations found in cfDNA was similar across races (p=0.31). However, receipt of genotype-matched therapy targeted to an actionable mutation varied by race, with the highest rates of matched therapy in White patients (White: 28%, Black: 11%, Hispanic 13%, Asian 14%, p&amp;lt; 0.001). After multivariable logistic regression adjusting for subtype, commercial insurance versus other insurance types, and proximity to the center, White patients remained significantly more likely to receive matched therapy (p=0.029). Conclusions: We observed significant race-based differences in non-English speaking status, insurance type, and median distance traveled to the institution. Racial/ethnic minority patients were less likely to receive genotype-matched therapy than White patients. Further research is needed to identify barriers and reduce disparities in access to precision medicine. Citation Format: Rupali Sood, Lianne Ryan, Andrzej Niemierko, Laura M. Spring, Dejan Juric, Steven J. Isakoff, Seth A. Wander, Jennifer Shin, Naomi Ko, Leif Ellisen, Beverly Moy, Aditya Bardia, Neelima Vidula. Impact of Race on Clinical, Socioeconomic, and Genomic Characteristics, Clinical Trial Participation, and Receipt of Genotype-matched Therapy Among Patients with Metastatic Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD1-10.

Timing of Diagnosis of Fetal Structural Abnormalities After the Introduction of Universal Cell-Free DNA in the Absence of First-Trimester Anatomical Screening

ABSTRACT Cell-free DNA (cfDNA) screens are now widely used for trisomy 21, 18, and 13 (and less often for sex-chromosome aberrations). In 2017, the Netherlands became the first country to implement genome-wide cfDNA as a first-tier screening test for chromosomal abnormalities. The use of this test for first-trimester screening subsequently increased from 32% to 45%, with 2% of women still choosing to have the combined test. Although the official purpose of these tests is for aneuploidy screening, studies demonstrate that at least one-fourth of structural abnormalities in fetuses can be detected during nuchal translucency (NT) measurement. Whether the routine addition of first-trimester anatomy screening (FTAS) would be effective in detecting severe fetal structural anomalies with low rates of false-positive results was felt to be important to address, to determine whether this should be an integral component of prenatal screening program. This study aimed to determine the gestational age at which fetal anomalies were detected and which anomalies detected by second-trimester anatomical screening could have theoretically been diagnosed via FTAS had this been performed on all women in the cohort. The data for this retrospective observational study were collected from pregnant women referred to the University Medical Center of Groningen Fetal Medicine Unit from 2017 through 2020. Women in the cohort were referred based on suspicion of fetal structural abnormalities following a dating ultrasound, or first- or second-trimester anatomy survey. First-trimester anatomy surveys were performed only in women with high-risk indications, including monochorionic twin pregnancy (or greater multiple pregnancy), first-degree relative of the fetus with structural abnormalities, more than 1 more distant relative with similar abnormalities, preexisting diabetes mellitus or first-trimester diagnosis of diabetes mellitus, high-titer thyroid-stimulating hormone receptor antibodies, or high radiation exposure of &gt;0.50 Gy. In the study, all information was collected regarding additional examinations, invasive testing, and pregnancy outcomes, including postpartum follow-up to confirm the diagnosis. Cases with an abnormal cfDNA result or increased risk for aneuploidy based on serum screening were not included, nor were “soft markers” for aneuploidy or variations of normal anatomy such as persistent left superior vena cava. In all, 547 pregnancies had second-trimester anomalies and were included in the analysis. A total of 13 pregnancies underwent FTAS, whereas the other 534 (97.6%) underwent only the dating ultrasound scan and second-trimester anatomy scanning. Average maternal age was 29 years, with a median body mass index of 25.1 kg/m2, and the mean gestational age at the time of the anatomical survey of 21.4 weeks. Of the 13 pregnancies that underwent FTAS, anomalies were confirmed in 12 fetuses, whereas 1 had edema that resolved. Overall, 492 fetuses (92.1%) had structural anomalies that were confirmed, and analysis was done to determine whether these would have been diagnosable in the first trimester. In all, 66 (13.4%) were classified as “always detectable” in the first trimester, 303 (61.6%) as “sometimes detectable,” and 123 (25.0%) as “never detectable.” The mean gestational age at diagnosis was 22.8 weeks. Limitations of the study include its hypothetical classification of abnormalities as “always,” “sometimes,” and “never,” based on the literature. However, the authors concluded that the introduction of an early anatomical survey together with noninvasive screening for aneuploidies is appropriate based on these results.

The accuracy of cell-free DNA screening for fetal segmental copy number variants: A systematic review and meta-analysis.

The performance of cell-free DNA (cfDNA) screening for microscopic copy number variants (CNVs) is unclear. This was a systematic review and meta-analysis to investigate the sensitivity, specificity and positive predictive value (PPV) of cfDNA screening for CNVs. Articles published in EMBASE, PubMed or Web of Science before November 2022 were screened for inclusion. This protocol was registered with PROSPERO (23 March 2021, CRD42021250849) prior to initiation. Articles published in English, detailing diagnostic outcomes for at least 10 high-risk CNV results with cfDNA were considered for inclusion. The PPV was calculated and pooled with random-effects models for double-arcsine transformed proportions, using cases with diagnostic confirmation. Overall sensitivity, specificity and a summary receiver-operating characteristics (ROC) curve were calculated using bivariate models. The risk of bias was assessed using QUADAS-2. In all, 63 articles were included in the final analysis, detailing 1591 459 cfDNA results. The pooled PPV was 37.5% (95% confidence interval [CI] 30.6-44.8), with substantial statistical heterogeneity (I2 = 93.9%). Bivariate meta-analysis estimated sensitivity and specificity to be 77.4% (95% CI 65.7-86.0) and 99.4% (95% CI 98.0-99.8), respectively, with an area under the summary ROC curve of 0.947 (95% CI 0.776-0.984). Approximately one-third of women who screen high-risk for CNVs with cfDNA will have an affected fetus. This value is of importance for screening counselling.

Multicenter clinical experience with non-invasive cell-free DNA screening for monosomy X and related X-chromosome variants.

We aimed to investigate how the presence of fetal anomalies and different X chromosome variants influences Cell-free DNA (cfDNA) screening results for monosomy X. From a multicenter retrospective survey on 673 pregnancies with prenatally suspected or confirmed Turner syndrome, we analyzed the subgroup for which prenatal cfDNA screening and karyotype results were available. A cfDNA screening result was defined as true positive (TP) when confirmatory testing showed 45,X or an X-chromosome variant. We had cfDNA results, karyotype, and phenotype data for 55 pregnancies. cfDNA results were high risk for monosomy X in 48/55, of which 23 were TP and 25 were false positive (FP). 32/48 high-risk cfDNA cases did not show fetal anomalies. Of these, 7 were TP. All were X-chromosome variants. All 16 fetuses with high-risk cfDNA result and ultrasound anomalies were TP. Of fetuses with abnormalities, those with 45,X more often had fetal hydrops/cystic hygroma, whereas those with "variant" karyotypes had different anomalies. Both, 45,X or X-chromosome variants can be detected after a high-risk cfDNA result for monosomy X. When there are fetal anomalies, the result is more likely a TP. In the absence of fetal anomalies, it is most often an FP or X-chromosome variant.

Prenatal diagnostic testing decisions in twin pregnancies in the era of cell-free DNA screening

Rates of diagnostic procedures in singleton pregnancies have declined since cell-free DNA (cfDNA) became commercially available. However, the impact of cfDNA on the uptake of diagnostic testing in twin gestations is unclear. We aimed to describe the rate of invasive diagnostic procedures in twin pregnancies after cfDNA screening with and without fetal structural abnormalities. This is a secondary analysis of data from a multicenter retrospective cohort study from 12/2011- 02/2020 that aimed to evaluate cfDNA screening performance in twin pregnancies. For this analysis, we excluded cases with a vanishing twin. cfDNA results were reported as positive, negative, or non-reportable. Structural abnormalities were defined as a major or minor anomaly, including an increased nuchal translucency >3mm. Rates of diagnostic testing were analyzed via chi-squared with odds ratio calculated by logistic regression. A total of 112 (6.6%) of 1,696 patients with twin pregnancies had cfDNA results and diagnostic procedures performed: 82 amniocenteses and 29 chorionic villus samplings (CVS). Diagnostic procedures were more frequent in patients that were older, had private insurance and conceived via IVF (Table 1). Patients with diagnostic testing were more likely to have positive or non-reportable cfDNA results compared to those without diagnostic testing. As noted in Figure 1A, 50% of positive cfDNA, 4.8% of negative cfDNA and 20% of non-reportable cfDNA ultimately had diagnostic testing. Figure 1B details the frequency of diagnostic procedures performed for each combination of cfDNA result and the presence of a structural abnormality. Diagnostic testing was more common if a structural abnormality was present (15.0% vs 4.5%, aOR 4.30 95% CI 2.71-6.83) and if there was an abnormal cfDNA result (32.8% vs 4.7%, aOR 8.72 95% CI 5.16-14.74), when adjusted for demographic factors. Cell-free DNA results and the presence of structural abnormalities greatly impact the decision to pursue diagnostic testing in patients with twin gestations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

380P Tumour naïve circulating tumour DNA (ctDNA) analysis in patients (pts) with resected colorectal cancer (CRC) in the phase III ASCOLT trial

Plasma ctDNA detects minimal residual disease (MRD) and is associated with disease recurrence in curatively treated CRC. In a randomised, double-blinded, phase III study (The ASpirin for Dukes C and high risk Dukes B COLorecTal cancer trial [ASCOLT], NCT00565708) evaluating the benefit of adjuvant aspirin, pts in the Australian and New Zealand cohort underwent prospective collection of plasma at baseline (within 90 days of completing adjuvant chemotherapy), 6 and 12 months, as well as surgical specimens. In a pilot cohort of 51 pts, we studied the utility of an ultra-sensitive ctDNA assay (SafeSEQ, Sysmex Inostics) for MRD detection. Cell-free DNA (cfDNA) analysis used the SafeSEQ CRC MRD assay (14 genes, AKT1, APC, BRAF, CTNNB1, ERBB3, FBXW7, KRAS, NRAS, PIK3CA, POLE, PPP2R1A, RNF43, SMAD4 and TP53). cfDNA results were correlated with recurrence data and tumour sequencing results using a 59-gene custom panel. All pts (26 female; median age 69; 16 high risk stage II, 35 III) received adjuvant 5-Fluorouracil based chemotherapy (32 with oxaliplatin, 4 had neoadjuvant chemoradiation for rectal cancer) and median follow-up was 60 months. 133 plasma samples were analysed (median 3.8 mL and DNA 71.0 genomic equivalents/ μL). Of the 10 pts with known recurrences (median time to recurrence 19 months), 6 had detectable cfDNA, including 2 of 3 pts without detectable tumour mutations (Table); median time from positive cfDNA to imaging detected recurrence was 28 weeks. Four pts with known recurrence did not have detectable cfDNA, including 1 who recurred 59 months after enrolment. Fourteen pts had plasma mutations without known recurrence. Matched peripheral blood mononuclear cell analyses are awaited.Table: 380PcfDNA mutationsMutant allele fraction (%, 1st sample if multiple)cfDNA detected at baselineAt 6mAt 12mConcordant mutation in tumourTime from positive cfDNA to recurrence on CT (w)Time from enrolment to recurrence (w)CEA elevated at CT recurrenceRecurrence site(s)BRAF V600ETP53 C275Y13.20.05Y- (Not done)-YN05YLiverSMAD4 R496C0.08NNYN4799YLiver, new primarynoneNNN257NPeritoneumKRAS G12VTP53 I195STP53 V272M0.040.160.04NYNYYYYYNYNN101101NLungKRAS G13DSMAD4 540PAPC R1399fs*9TP53 R342*8.42.42.56.5NNNNNNNNYYYYYYNN048NLung, nodalnoneNNN80NLung, nodalTP53 Y220C0.03-Y-N7198YNodal, localKRAS G12VTP53 R306*0.040.07NYYN-YN024NLungnoneNN-30NLungnoneNNN87NLung Open table in a new tab In 51 ASCOLT pts with high risk resected CRC following adjuvant chemotherapy, 6 of 10 with reported recurrences had detectable cfDNA using a tumour naïve SafeSEQ assay. Analyses in the larger cohort (n = 368) are underway.