cfDNA Testing Research Articles

P–560 Comparative analysis of non-invasive preimplantation genetic testing of aneuploidies (niPGT-A), PGT-A and IVF cycles without aneuploidy testing: preliminary results

Abstract Study question Can non-invasive preimplantation genetic testing of aneuploidies (niPGT-A) improve the clinical outcome in IVF patients after proper validation? Summary answer We demonstrate the usefulness of the embryonic cell-free DNA (cfDNA) in the blastocyst culture medium to select more objectively the blastocysts with higher implantation potential. What is known already One of the greatest challenges in IVF is accurately selecting viable embryos that are more likely to achieve healthy livebirths following embryo transfer. Trophectoderm (TE) biopsy and PGT-A provide a direct assessment of chromosome status and improve implantation and clinical pregnancy rates per transfer. A non-invasive alternative is to analyse embryonic cfDNA in the blastocyst culture medium. Previous studies have shown that cfDNA testing in culture medium of blastocysts on day 6 of development allows aneuploidy detection with high concordance rates compared to TE biopsy and inner cell mass (Rubio et al., 2020). Study design, size, duration Observational study of the clinical application of niPGT-A (July 2020-December 2020). The clinical application consisted in a first validation phase, comparing TE biopsies with cfDNA in the media of 28 blastocysts. And, in a second phase, niPGT-A was applied and the outcome of 13 single embryo transfers (SETs) compared to 13 PGT-A SETs and 130 IVF/ICSI SETs performed in a period of six months. In the three groups, women and donors age was ≤38 years. Participants/materials, setting, methods Embryos were cultured in a Geri incubator (Merck) up to day 4, and then individually cultured in 10µl drops of CCSS (Fujifilm) until day 6 in a bench-top K-system. At day 6, blastocysts were vitrified, and media collected in sterile PCR tubes after at least 40 hours in culture. After collection, media were immediately frozen and analyzed by NGS analysis in our reference laboratory (Igenomix, Spain). Deferred transfer was performed according to media results. Main results and the role of chance Before the first clinical cases, a validation of the protocol comparing the results of cfDNA with the TE biopsies of the same day–6 blastocyst was performed, and ploidy concordance rates were 87.5%. Similar results were found for niPGT-A and PGT-A in terms of aneuploidy results and in clinical outcomes. The percentages of informative results were 95% and 97% and the aneuploidy rates were 44% and 46%, for niPGT-A and PGT-A, respectively. Clinical pregnancy rates were in both groups of aneuploidy testing, 69.2%, with 8 ongoing pregnancies (61.5%) and 4 tested by prenatal screaning NACE. For untested embryos clinical pregnancy (57.7%) and ongoing pregnancy rates (48.5%) were lower than in the two groups of tested embryos (niPGT-A and PGT-A). In the niPGT-A cycles embryo transfer was performed according to media results and morphology. We did a secondary analysis of which blastocyst we would transfer, if only morphology is considered. We observed that if we only select the embryos by morphology, in 61.5% of the cases we would choose the same embryo than with niPGT-A, and in 30.4% of the cases we would transfer a blastocyst with an aneuploid medium. Limitations, reasons for caution Our results are encouraging but should be interpreted with caution due to the small sample size. Larger and randomized controlled trials are needed to verify and extend our findings in each group. Wider implications of the findings: We observed consistent results for niPGT-A compared to TE biopsies in our internal validation. These results endorse the clinical application of niPGT-A in the routine of the laboratory and can avoid the embryo manipulation also reducing the subjectivity when embryos are selected only by morphology. Trial registration number Sa–16552/19-EC:428

Validation of a Circulating Tumor DNA-Based Next-Generation Sequencing Assay in a Cohort of Patients with Solid tumors: A Proposed Solution for Decentralized Plasma Testing.

BackgroundCharacterization of circulating tumor DNA (ctDNA) has been integrated into clinical practice. Although labs have standardized validation procedures to develop single locus tests, the efficacy of on‐site plasma‐based next‐generation sequencing (NGS) assays still needs to be proved.Materials and MethodsIn this retrospective study, we profiled DNA from matched tissue and plasma samples from 75 patients with cancer. We applied an NGS test that detects clinically relevant alterations in 33 genes and microsatellite instability (MSI) to analyze plasma cell‐free DNA (cfDNA).ResultsThe concordance between alterations detected in both tissue and plasma samples was higher in patients with metastatic disease. The NGS test detected 77% of sequence alterations, amplifications, and fusions that were found in metastatic samples compared with 45% of those alterations found in the primary tumor samples (p = .00005). There was 87% agreement on MSI status between the NGS test and tumor tissue results. In three patients, MSI‐high ctDNA correlated with response to immunotherapy. In addition, the NGS test revealed an FGFR2 amplification that was not detected in tumor tissue from a patient with metastatic gastric cancer, emphasizing the importance of profiling plasma samples in patients with advanced cancer.ConclusionOur validation experience of a plasma‐based NGS assay advances current knowledge about translating cfDNA testing into clinical practice and supports the application of plasma assays in the management of oncology patients with metastatic disease. With an in‐house method that minimizes the need for invasive procedures, on‐site cfDNA testing supplements tissue biopsy to guide precision therapy and is entitled to become a routine practice.Implications for PracticeThis study proposes a solution for decentralized liquid biopsy testing based on validation of a next‐generation sequencing (NGS) test that detects four classes of genomic alterations in blood: sequence mutations (single nucleotide substitutions or insertions and deletions), fusions, amplifications, and microsatellite instability (MSI). Although there are reference labs that perform single‐site comprehensive liquid biopsy testing, the targeted assay this study validated can be established locally in any lab with capacity to offer clinical molecular pathology assays. To the authors' knowledge, this is the first report that validates evaluating an on‐site plasma‐based NGS test that detects the MSI status along with common sequence alterations encountered in solid tumors.

Performance of a universal prenatal screening program incorporating cell-free fetal DNA analysis in Ontario, Canada.

BACKGROUND:The emergence of cell-free fetal DNA (cfDNA) testing technology has disrupted the landscape of prenatal screening for trisomies 21 (T21) and 18 (T18). Publicly funded systems around the world are grappling with how to best integrate this more accurate but costly technology, as there is limited evidence about its incremental value in real-world conditions. The objectives of this study were to describe the population-based performance of Ontario’s prenatal screening program, which incorporates publicly funded cfDNA screening for specific indications, and the effect of cfDNA testing on the screening and diagnostic choices made by pregnant people.METHODS:We conducted a retrospective, descriptive cohort study using routinely collected data from Better Outcomes & Registry Network (BORN) Ontario, which captures linked population data for prenatal and neonatal health encounters across Ontario. We included all singleton pregnancies with an estimated due date between Sept. 1, 2016, and Mar. 31, 2019, that underwent publicly funded prenatal screening in Ontario, and a comparison cohort from Apr. 1, 2012, and Mar. 31, 2013. We assessed performance of the screening program for the detection of T21 or T18 by calculating sensitivity, specificity, positive predictive value and negative predictive value against diagnostic cytogenetic results or birth outcomes. We assessed the impact of the program by calculating the proportion of T21 screen-positive pregnancies undergoing subsequent cfDNA screening and invasive prenatal diagnostic testing.RESULTS:The study cohort included 373 682 pregnancies. The prenatal screening program had an uptake of 69.9%, a screen-positive rate and sensitivity of 1.6% and 89.9% for T21, and 0.2% and 80.5% for T18, respectively. The test failure rate for cfDNA screening was 2.2%. Invasive prenatal diagnostic testing decreased from 4.4% in 2012–2013 to 2.4% over the study period; 65.2% of pregnant people who received a screen-positive result from cfDNA testing went on to have invasive prenatal diagnostic testing.INTERPRETATION:This publicly funded screening program, incorporating cfDNA analysis for common aneuploidies, showed robust performance, a substantial reduction in invasive prenatal diagnostic testing and that pregnant people exercise autonomy in their choices about prenatal screening and diagnosis.

Technical Evaluation of the COBAS EGFR Semiquantitative Index (SQI) for Plasma cfDNA Testing in NSCLC Patients with EGFR Exon 19 Deletions

The cobas® EGFR Test provides a semiquantitative index (SQI) that reflects the proportion of mutated versus wild-type copies of the EGFR gene in plasma. The significance of SQI as an indirect measure of the variant allele frequency (VAF) or mutated copies/mL remains unclear. The aim of this study was to evaluate the correlation of SQI with the VAF and the number of mutated copies/mL obtained by a digital droplet PCR (ddPCR) test in NSCLC samples. The study included 118 plasma samples from a retrospective cohort of 25 stage IV adenocarcinoma patients with EGFR exon 19 deletions (Ex19Del), obtained before and during tyrosine kinase inhibitor (TKI) treatment. Both SQI and VAF and SQI and mutated copies/mL showed the same significant correlation (r2 = 0.79, p < 0.00001) across the whole study cohort. We found better correlation in samples collected at the baseline between SQI and VAF (r2 = 0.94, p < 0.00001) and SQI and mutated copies/mL (r2 = 0.97, p < 0.00001) compared to samples collected during TKI treatment: r2 = 0.76; p < 0.00001 for SQI and VAF and r2 = 0.75; p < 0.00001 for SQI and mutated copies/mL. The study indicates that SQI is a robust quantitative indirect measure of VAF and the number of mutated copies/mL in plasma from patients with an EGFR Ex19Del mutation. Further studies are desirable to assess the SQI cut-off values related to the clinical status of the patient.

Characterization and Clinical Utility of BRAFV600 Mutation Detection Using Cell-Free DNA in Patients with Advanced Melanoma.

Simple SummaryThe choice of cancer drug(s) for the treatment of advanced melanoma is based on the types of gene alterations that are present in the patient’s tumor(s). Sometimes, the tumor sample that is obtained from surgery may be degraded, and the test does not provide a reliable result, leading to the selection of the wrong treatment, and, consequently, poor outcomes for the patient. Surgery to obtain fresh tumor samples is inconvenient. In recent years, scientists have learned that fragments of genes from dying cells, including tumors, are constantly being released into the blood. This study shows that the presence of altered genes can be reliably determined using easy-to-obtain blood samples. The study also shows that, while there is a small rate of error with the commonly used tests based on the tumor tissue sample, retests using blood samples may be a less invasive and rapid alternative for identifying the BRAF mutation status and selecting the right treatment for these patients.Tissue-based tests for BRAFV600 mutation-positive melanoma involve invasive biopsy procedures, and can lead to an erroneous diagnosis when the tumor samples degrade. Herein, we explored a minimally invasive, cell-free deoxyribonucleic acid (cfDNA)-based platform, to retest patients for BRAFV600 mutations. This phase 2 study enrolled adult patients with unresectable/metastatic melanoma. A prescreening testing phase evaluated the concordance between a prior tissue-based BRAFV600 mutation test result and a subsequent plasma cfDNA-based test result. A treatment phase evaluated the patients who were confirmed as BRAFV600 mutation-positive, and were treated with cobimetinib plus vemurafenib. It was found that 35/54 patients (64.8%) with a mutant BRAF status by prior tissue test had a positive BRAFV600 mutation with the cfDNA test. Further, 7/118 patients (5.9%) with a wild-type BRAF status had a positive BRAFV600 mutation cfDNA test; tissue retests on archival samples confirmed BRAFV600 mutation positivity in 5/7 patients (71.4%). One of these patients received BRAF pathway-targeted therapy (cobimetinib plus vemurafenib), and had progression-free survival commensurate with previous experience. In the overall cobimetinib plus vemurafenib-treated population, 29/36 patients (80.6%) had an objective response. The median progression-free survival was 13.6 months (95% confidence interval, 9.5–16.5). Cell-free DNA–based tests may be a fast and convenient option to identify BRAF mutation status in melanoma patients, and help inform treatment decisions.

Cell‐free DNA testing of maternal blood in screening for trisomies in twin pregnancy: updated cohort study at 10–14 weeks and meta‐analysis

ABSTRACTObjectiveTo expand the limited knowledge on cell‐free DNA (cfDNA) analysis of maternal blood for trisomies 21, 18 and 13 in twin pregnancy by updating the data from The Fetal Medicine Foundation (FMF) on prospective first‐trimester screening and those identified in a systematic review of the literature.MethodsThe FMF data were derived from prospective screening for trisomies 21, 18 and 13 in twin pregnancies at 10 + 0 to 14 + 1 weeks' gestation using the Harmony® prenatal test. A search of MEDLINE, EMBASE, CENTRAL (The Cochrane Library), ClinicalTrials.gov and the International Clinical Trials Registry Platform (World Health Organization) was carried out to identify all peer‐reviewed publications on clinical validation or implementation of maternal cfDNA testing for trisomies 21, 18 and 13 in twin pregnancy, irrespective of gestational age at testing, in which data on pregnancy outcome were provided for at least 85% of the study population. Meta‐analysis was performed using the FMF data and data from the studies identified by the literature search. This review was registered in the PROSPERO international database for systematic reviewsResultsIn the FMF study, cfDNA testing was carried out in 1442 twin pregnancies and a result was obtained, after first or second sampling, in 1367 (94.8%) cases. In 93.1% (1272/1367) of cases, there was prenatal or postnatal karyotyping or birth of phenotypically normal babies; 95 cases were excluded from further analysis either because the pregnancy ended in termination, miscarriage or stillbirth with no known karyotype (n = 56) or there was loss to follow‐up (n = 39). In the 1272 pregnancies included in the study, there were 20 cases with trisomy 21, 10 with trisomy 18, two with trisomy 13 and 1240 without trisomy 21, 18 or 13. The cfDNA test classified correctly 19 (95.0%) of the 20 cases of trisomy 21, nine (90.0%) of the 10 cases of trisomy 18, one (50.0%) of the two cases of trisomy 13 and 1235 (99.6%) of the 1240 cases without any of the three trisomies. The literature search identified 12 relevant studies, excluding our papers because their data are included in the current study. In the combined populations of our study and the 12 studies identified by the literature search, there were 137 trisomy‐21 and 7507 non‐trisomy‐21 twin pregnancies; the pooled weighted detection rate (DR) and false‐positive rate (FPR) were 99.0% (95% CI, 92.0–99.9%) and 0.02% (95% CI, 0.001–0.43%), respectively. In the combined total of 50 cases of trisomy 18 and 6840 non‐trisomy‐18 pregnancies, the pooled weighted DR and FPR were 92.8% (95% CI, 77.6–98.0%) and 0.01% (95% CI, 0.00–0.44%), respectively. In the combined total of 11 cases of trisomy 13 and 6290 non‐trisomy‐13 pregnancies, the pooled weighted DR and FPR were 94.7% (95% CI, 9.14–99.97%) and 0.10% (95% CI, 0.03–0.39%), respectively.ConclusionsIn twin pregnancy, the reported DR of trisomy 21 by cfDNA testing is high, but lower than that in singleton pregnancy, whereas the FPR appears to be equally low. The number of cases of trisomy 18 and more so trisomy 13 was too small for accurate assessment of the predictive performance of the cfDNA test. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Contingent prenatal screening for frequent aneuploidies with cell-free fetal DNA analysis

ObjectiveTo analyze the results of contingent screening for common aneuploidies at our center from June 2017 to June 2019. Materials and methodsTraditional screening tests were performed using a combination of biochemical markers and ultrasound measurements in the first and second trimesters to assess the risk of trisomies 21 (T21), 18 (T18) and 13 (T13). Cell-free DNA (cf-DNA) testing was offered (Harmony test) to pregnant women at high risk (>1/280 for T21 and > 1/150 for T13 and T18) and a normal early morphology scan. In positive cases, prenatal sampling was strongly recommended to confirm the results by gold standard methods (QF-PCR and karyotyping). Newborns' phenotypes were corroborated after birth in all cases. ResultsIn this prospective study, 8153 pregnant women were enrolled, resulting in 390 at high risk according to traditional screening tests. cfDNA testing was offered to 383 women. Traditional screening tests showed a false negative rate of 9.68% for T21. Traditional test sensitivity for T21 was 90.3%, for a false positive rate of 4.17% and a positive predictive value of 7.6%. The positive and negative predictive value for cfDNA testing was 100%. The approach used avoided invasive procedures in 91.3% of women at high risk. The prevalence of chromosomal abnormalities in the population analyzed was 1 in 164, and 1 in 210 for T21. ConclusionsOur results show that offering cf-DNA testing to women at high risk in traditional tests (including those with risks >1 in 50) significantly reduces false positives and, therefore, the number of invasive tests. Extending the use of cf-DNA testing to intermediate risk categories may be cost effective.

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

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

Liquid Biopsy As a Diagnostic Test for Myelodysplastic Syndrome and Other Acute and Chronic Myeloid Neoplasms

Cytopenia is common in cause for considering the diagnosis of myelodysplastic syndrome (MDS), especially in the elderly population. To diagnose the presence or absence of MDS, bone marrow biopsy is performed on most patients with cytopenia. Performing bone marrow biopsy is a painful procedure and can be associated with bleeding and other complications. Moreover, bone marrow morphology is unreliable for the diagnosis of MDS. Cytogenetics and molecular studies can provide conclusive evidence for the diagnosis of MDS. Liquid biopsy and testing for molecular abnormalities in cfDNA in peripheral blood plasma is significantly less invasive than traditional bone marrow testing. We evaluated the diagnostic value of liquid biopsy in determining the presence of MDS in 640 patients presented in community setting practice with cytopenia.Methodology: A total of 640 peripheral blood samples from patients with cytopenia were submitted for cfDNA testing. We used the TruSight Myeloid panel (Illumina, San Diego, CA) for detecting missense mutations and fragment length analysis (FLA) for detecting ITD in FLT3 and large indels in CALR . DNA was extracted from samples using the QIAamp DNA Mini Kit. This next generation sequencing (NGS) testing covers mutations in 54 myeloid-related genes. The average depth of sequencing was 10,000x.Results: Of the 640 tested patients, 310 (48.4%) showed mutations in one or more genes: 38% of these had mutations in one gene, 25.5% had mutations in two genes, and 36.5% had mutations in more than two genes. However, 14% of cases with mutation in one gene had two or more mutant subclones. The median age of all patients was 69 (range: 15-99) and 55% were males. Patients with mutations were significantly older (75 vs. 66 years old, P<0.0001) and more likely to be males (60% vs. 46% males, P=0.004). The most commonly mutated genes were TET2, DNMT3A, ASXL1, SRSF2, SF3B1, and RUNX1 detected in 31%, 28%, 25%, 17%, 15%, and 10% of the patients with mutations, respectively. Interestingly, 32 patients (10% of patients with any mutation) had mutations in TP53, 3% had mutations in FLT3, and 4% had mutations in NPM1 genes, indicating aggressive or acute disease. The average variant allele frequency (VAF) was <10% in 22% of cases with any mutation, between 10-20% in 19% of cases, between 20-30% in 16% of cases, and >40% in 43% of the cases. There was a significantly higher number of mutated genes in patients with average VAF >20% (P<0.0001). The combination of VAF, type of mutated gene, and number of mutated genes were considered in determining the significance of abnormal neoplastic clone and recommendation for management. The presence of mutations in NPM1, FLT3, TP53 as well as SF3B1 were considered diagnostic irrespective of the VAF. For patients with VAF at <20% in genes such as TET2, DNMT3A, and ASXL1 were not considered diagnostic for clinically significant myeloid neoplasm.Conclusions: Liquid biopsy for evaluating patients with pancytopenia can be used to evaluate the presence of abnormal hematopoietic neoplasm clones and the severity of the disease. Liquid biopsy can reduce the need for bone marrow biopsy in more than 50% of patients presenting with cytopenia. VAF, mutated gene types, and the combination of mutated genes should be considered for making management decisions based on liquid biopsy. [Display omitted] DisclosuresMa:NeoGenomics: Employment. De Dios:NeoGenomics: Employment. Funari:NeoGenomics: Employment. Jiang:NeoGenomics: Employment. Agersborg:NeoGenomics: Employment. Hummel:NeoGenomics: Employment. Blocker:NeoGenomics: Employment. Albitar:NeoGenomics: Employment.

Diagnostic accuracy of Mycobacterium tuberculosis cell-free DNA for tuberculosis: A systematic review and meta-analysis.

BackgroundDiagnosis of tuberculosis (TB) is still difficult. The purpose of our study was to evaluate the diagnostic accuracy of Mycobacterium tuberculosis cell-free DNA (cfDNA) for diagnosing of TB.MethodsWe searched relevant databases for studies that used cfDNA to diagnose TB. We evaluated the accuracy of cfDNA compared with the composite reference standard (CRS) and culture. True positive, false positive, false negative, and true negative values for cfDNA were obtained first, then the estimated pooled sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), diagnostic odds ratio (DOR), and the area under the summary receiver operating characteristic (SROC) curve (AUC) of cfDNA for diagnosing TB were calculated with 95% confidence intervals (CIs). Heterogeneity was determined using the I2 statistic. When the heterogeneity was obvious, the source of heterogeneity was further discussed.ResultsWe included 14 independent studies comparing cfDNA with the CRS, and 4 studies compared with culture. The pooled sensitivity, specificity, PPV, NPV, DOR, and AUC of the SROC were 68%, 98%,99%, 62%, 83, and 0.97 as compared with the CRS, respectively. The pooled sensitivity, specificity, PPV, NPV, DOR, and AUC of the SROC were 48%, 91%, 92%, 60%, 5, and 0.88 as compared with culture, respectively. The heterogeneity between studies was significant.ConclusionsThe accuracy of cfDNA testing for TB diagnosis was good compared with CRS and culture. cfDNA can be used for rapid early diagnosis of TB.

EPCT-21. NEXT-GENERATION SEQUENCING OF CEREBROSPINAL FLUID FOR CLINICAL MOLECULAR DIAGNOSTICS IN ADOLESCENT AND YOUNG ADULT (AYA) BRAIN TUMOR PATIENTS

PurposePediatric central nervous system tumors remain a leading cause of cancer-related death in children and adolescents. Safe sampling of tumor tissue for diagnostic purposes may be challenging. Subclinical detection of disease prior to clinical or imaging progression may provide opportunity for earlier intervention and ultimately improve overall survival. Additionally, our understanding of molecular evolution in response to therapy remains limited, given the rarity of serial sampling of tumor tissue. MethodsWe report our experience with minimally invasive molecular diagnostics using a validated next generation sequencing assay for sequencing of cerebrospinal fluid (CSF) cell-free DNA (cfDNA) obtained at the time of surgery, by intraventricular catheter or lumbar puncture. All CSF samples were collected as part of clinical care, and results reported to both clinicians and patients/families. ResultsWe analyzed 64 CSF samples from 45 pediatric and adolescent and young adult (AYA) patients (pediatric=25; AYA=20) with primary and recurrent brain tumors across 12 histopathological subtypes including high-grade glioma (n=10), medulloblastoma (n=10), pineoblastoma (n=5), low grade glioma (n=4), diffuse leptomeningeal glioneuronal tumor (DLGNT) (n=4), metastatic retinoblastoma (n=4), ependymoma (n=3), and other (n=5). Somatic alterations were detected in 28/64 samples (44.4%) and in at least one sample per unique patient in 22/45 patients (48.8%). CSF cfDNA positivity was strongly associated with the presence of disseminated disease at the time of collection (86.3%). No association was seen between CSF cfDNA positivity and the timing of CSF collection during the patient’s disease course. ConclusionWe identified four general categories where CSF cfDNA testing provided additional relevant diagnostic, prognostic, and/or therapeutic information, impacting clinical assessment and decision making: 1) diagnosis; 2) identification of actionable alterations; 3) track response to therapy; and 4) monitoring tumor evolution. Our findings support broader implementation of clinical CSF cfDNA testing in this population that may improve care.

Economic impact of using maternal plasma cell-free DNA testing to guide further workup in recurrent pregnancy loss.

ObjectiveWe have previously demonstrated that maternal‐plasma cell‐free DNA (cfDNA)‐testing can detect chromosomal anomalies in recurrent pregnancy loss (RPL) with 81.8% sensitivity and 90.3% specificity. Here we assess whether this is cost effective in guiding further workup in RPLs.MethodA decision‐analytic model was developed to compare the cost of various RPL management pathways: (1) current American Society for Reproductive Medicine (ASRM) RPL workup; (2) microarray or karyotyping analysis of products of conception (POCs) and RPL workup only for euploid cases; and (3) cfDNA testing and RPL workup only for euploid cases. Sample accessibility, failure rates, and sensitivity were specified for each test. Costs of sample collection, genetic tests, and RPL workup were considered. Analysis outcomes included detection rate of chromosomal anomaly and cost per patient tested.ResultsIn comparison to existing cytogenetic testing on POCs, cfDNA testing pathway allowed for better sample accessibility with a lower cost per patient. In addition, using cfDNA to guide further workup significantly increases the number of causative fetal chromosome anomalies detected, reducing the number of patients undergoing unnecessary workup resulting in an overall cost savings.ConclusionOur study showed that inclusion of cfDNA testing is a cost‐effective approach to guide RPL workup.

Genomic landscape of cell-free DNA in a diverse cohort of metastatic breast cancer patients.

e13066 Background: Breast cancer is the most prevalent cancer and the second leading cause of cancer deaths among women in the United States. Once the primary breast cancer has metastasized to other parts of the body, despite treatment, patients have an incurable disease. Compared to the primary tumor, often the metastasized tumor sites have different mutational profiles that are not represented in the initial biopsy. Liquid biopsy is becoming a more commonly accepted method in clinical practice to determine the heterogeneity of metastatic tumors by analyzing cell-free DNA (cfDNA). Several studies have demonstrated that cfDNA can be utilized as an effective prognostic tool and can reveal multiple clinically targetable alterations in metastatic breast cancer (mBC). Methods: We characterized the genomic landscape of cfDNA in a diverse patient population and across different subtypes of mBC. We performed a retrospective chart review in patients (n = 41) with metastatic breast cancer from a medical oncology clinic within the Hawaii Pacific Health system. Patients who had at least one cfDNA testing by Guardant360 were evaluated. Patient demographics, age at diagnosis, race, subtype and mutations were reviewed along with treatment information. Results: We found no statistically significant differences in genomic landscape according to race, however there were differences seen in tumor genomics according to age and subtype. Older patients were less likely to have detectable disease on liquid biopsy compared to younger patients (age 70+ and 60-69 vs 30-39; xp 0.02). Consistent with subtype, mutations in ESR1 (n = 12) and PIK3CA (n = 14) were more commonly seen in hormone positive (HR+) mBC where known tailored treatment options are available (ie. fulvestrant and alpelisib respectively). Postmenopausal patients were more likely to have ESR1 mutations than premenopausal patients (50-59 and 70+ vs 30-39 and 40-49, xp 0.009). About half (n = 20) had TP53 mutations, followed by PIK3CA (n = 14), ESR1 (n = 12), CCND1 (n = 7) and MYC (n = 6). Conclusions: We evaluated 41 unique cases of mBC and correlated them with their genomic profiles. We plan to further investigate genomic changes according to treatment with subsequent repeat liquid biopsies. We hope to utilize these findings to help prognosticate and guide future treatment options.

Sputum cell-free DNA: Valued surrogate sample for the detection of EGFR exon 20 p.T790M mutation in patients with advanced lung adenocarcinoma and acquired resistance to EGFR-TKIs.

BackgroundSputum cell‐free DNA (cfDNA) is a valuable surrogate sample for assessing EGFR‐sensitizing mutations in patients with advanced lung adenocarcinoma. Detecting EGFR exon 20 p.T790 M (p.T790 M) is much more challenging due to its limited availability in tumor tissues. Exploring sputum cfDNA as an alternative for liquid‐based sample type in detecting p.T790 M requires potential improvement in clinical practice.MethodsA total of 34 patients with EGFR‐sensitive mutation‐positive lung adenocarcinoma and acquired resistance to the first generation of epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) were enrolled. The sputum samples, and paired tumors and/or plasma samples were tested for p.T790 M mutation and concordance of p.T790 M status among the three sample types was analyzed.ResultsThe overall concordance rate of p.T790 M mutation between sputum cfDNA and tumor tissue samples was 85.7%, with a sensitivity of 66.7% and a specificity of 100%. The sensitivity for detecting p.T790 M in sputum cfDNA was 100%, 66.7%, and 0% in the three sputum groups of malignant, satisfactory but no malignant cells, and unsatisfactory, respectively. The combined results of plasma cfDNA testing and sputum cfDNA testing further increased the sensitivity to 100% for p.T790 M detection in satisfactory but no malignant cells sputum group.ConclusionThese findings revealed that cfDNA from malignant or satisfied but no malignant cells sputum is considered suitable for detecting p.T790 M mutation in patients with acquired resistance to first or second‐generation EGFR‐TKIs. The sputum cytological pathological evaluation‐guided sputum cfDNA testing assists in significantly improving the sensitivity of p.T790 M detection, bringing significant value for the maximal application of third‐generation EGFR‐TKIs in second‐line treatment.

Clinical Utility of Circulating Cell-Free DNA Mutations in Anaplastic Thyroid Carcinoma.

Background: Anaplastic thyroid carcinoma (ATC) is an aggressive thyroid cancer that requires a rapid diagnosis and treatment to achieve disease control. Gene mutation profiling of circulating cell-free DNA (cfDNA) in peripheral blood may help to facilitate early diagnosis and treatment selection. The relatively rapid turnaround time compared with conventional tumor mutation testing is a major advantage. The objectives of this study were to examine the concordance of ATC-related mutations detected in cfDNA with those detected in the corresponding tumor tissue, and to determine the prognostic significance of cfDNA mutations in ATC patients. Methods: The ATC patients who were diagnosed and treated at The University of Texas MD Anderson Cancer Center between January 2015 and February 2018 and who had cfDNA testing were included in this study. cfDNA was collected by blood draw and was analyzed by next-generation sequencing (NGS) using the Guardant360-73 gene platform. Results: A total of 87 patients were included in the study. The most frequently mutated genes detected in cfDNA were TP53, BRAF, and PIK3CA. In 28 treatment naive ATC patients, the concordance rate of detected mutations in TP53, BRAFV600E, and PIK3CA between cfDNA and matched tissue NGS was 82.1%, 92.9%, and 92.9%, respectively. Patients with a PIK3CA mutation detected on cfDNA had worse overall survival (OS) (p = 0.03). This association was observed across various treatment modalities, including surgery, cytotoxic chemotherapy, radiation, and BRAF inhibitor (BRAFi) therapy. With regard to treatment, BRAFi therapy significantly improved ATC OS (p = 0.003). Conclusions: cfDNA is a valuable tool to evaluate a tumor's molecular profile in ATC patients. We identified high concordance rates between the gene mutations identified via cfDNA analysis and those identified from the NGS of the corresponding tumor tissue sequencing. Identified mutations in cfDNA can potentially provide timely information to guide treatment selection and evaluate the prognosis in patients with ATC.

Next generation sequencing for pathogen detection in periprosthetic joint infections.

Periprosthetic joint infections (PJI) represent one of the most catastrophic complications following total joint arthroplasty (TJA). The lack of standardized diagnostic tests and protocols for PJI is a challenge for arthroplasty surgeons.Next generation sequencing (NGS) is an innovative diagnostic tool that can sequence microbial deoxyribonucleic acids (DNA) from a synovial fluid sample: all DNA present in a specimen is sequenced in parallel, generating millions of reads. It has been shown to be extremely useful in a culture-negative PJI setting.Metagenomic NGS (mNGS) allows for universal pathogen detection, regardless of microbe type, in a 24–48-hour timeframe: in its nanopore-base variation, mNGS also allows for antimicrobial resistance characterization.Cell-free DNA (cfDNA) NGS, characterized by lack of the cell lysis step, has a fast run-time (hours) and, together with a high sensitivity and specificity in microorganism isolation, may provide information on the presence of antimicrobial resistance genes.Metagenomics and cfDNA testing have reduced the time needed to detect infecting bacteria and represent very promising technologies for fast PJI diagnosis.NGS technologies are revolutionary methods that could disrupt the diagnostic paradigm of PJI, but a comprehensive collection of clinical evidence is still needed before they become widely used diagnostic tools.Cite this article: EFORT Open Rev 2021;6:236-244. DOI: 10.1302/2058-5241.6.200099

Adoption of a non-invasive prenatal test (NIPT) in prenatal screening in Moscow: first results

The objective — To assess the effectiveness of including NIPT in the structure of prenatal diagnostics in Moscow. Material and Methods — Totally 5,181 pregnancies undergoing screening for fetal trisomy using NIPT during the period from 01.04.2020 to 30.09.2020 in Russia. According to the results of biochemical blood test, the patients were divided into two groups: group of high risk (cut-off ≥1:100) (n=208) and group of intermediate risk (cut-off 1:101 – 1:2500) (n=4,973). Patients at high-risk cell-free DNA (cfDNA) were offered an invasive procedure, followed by genetic analysis (cytogenetic or molecular karyotyping). Results — Among the analysed samples, 117 (2.3%) had a high risk of the following common fetal chromosome abnormalities by NIPT: trisomy 21 in 50 cases, trisomy 18 in 17 cases, trisomy 13 in 5 cases, and sex chromosome aneuploidy (SCA) in 22 cases. Additionally, rare autosomal trisomies and/or subchromosomal arrangements were revealed in 23 cases. We found associations between cfDNA concentration and high risk of aneuploidies (particularly trisomy 21) and fetal sex and between low fetal fraction (FF) and body mass index (BMI) as well as maternal weight. Additionally, a high risk of trisomy 21 was associated with the term gestation. Conclusion — The effectiveness of technological resources that are based on cfDNA testing for detecting abnormal fetal chromosome numbers and other chromosomal anomalies is high and reduce rates of false positive results. Therefore, NIPT should be more widely used as a first-line screening method.

Cell‐free DNA analysis of maternal blood in prenatal screening for chromosomal microdeletions and microduplications: a systematic review

Scientific Societies do not recommend the use of cell-free DNA (cfDNA) testing as a first-tier screening for microdeletion and microduplication syndromes (MMs). The aim of this study was to review the current available literature on the performance of cell-free DNA as a screening for MMs. Medline, Embase and the Cochrane Library were searched electronically from 2000 to January 2020 and articles reporting the diagnostic performance of cfDNA screening for MMs in large (>5000 cases) series were included. Between-study heterogeneity and random effect model for screen positive rate (SPR), false positive rate (FPR) and positive predictive value (PPV) were calculated. We identified 42 papers, seven included, for a total of 474,189 pregnancies and 210 cases of MMs. Diagnostic verification of positive cases was available overall in 486 (71.68 %) of 678 cases. The weighted pooled SPR, FPR and PPV were 0.19% (95% CI=0.09-0.33), 0.07 (95% CI=0.02-0.15) and 44.1 (95% CI=31.49-63.07). In conclusion, the pooled PPV of cfDNA testing in screening for MMs was about 40%, ranging from 29% to 91%, for an overall FPR <0.1%. No confirmatory analysis was available in cases that did not undergo invasive testing, which were the vast majority of cases with a negative test, and therefore, the DR and the negative predictive value cannot be determined.

Cell-Free DNA for the Management of Classical Hodgkin Lymphoma.

Cell-free DNA (cfDNA) testing, is an emerging “liquid biopsy” tool for noninvasive lymphoma detection, and an increased amount of data are now available to use this technique with accuracy, especially in classical Hodgkin lymphoma (cHL). The advantages of cfDNA include simplicity of repeated blood sample acquisition over time; dynamic, noninvasive, and quantitative analysis; fast turnover time; reasonable cost; and established consistency with results from tumor genomic DNA. cfDNA analysis offers an easy method for genotyping the overall molecular landscape of pediatric and adult cHL and may help in cases of diagnostic difficulties between cHL and other lymphomas. cfDNA levels are correlated with clinical, prognostic, and metabolic features, and may serve as a therapeutic response evaluation tool and as a minimal residual disease (MRD) biomarker in complement to positron emission tomography (PET). Indeed, cfDNA real-time monitoring by fast high-throughput techniques enables the prompt detection of refractory disease or may help to address PET residual hypermetabolic situations during or at the end of treatment. The major recent works presented and described here demonstrated the clinically meaningful applicability of cfDNA testing in diagnostic and theranostic settings, but also in disease risk assessment, therapeutic molecular response, and monitoring of cHL treatments.

P89.08 Real-World Impact of Plasma Cell-Free DNA Next-Generation Sequencing to Detect Actionable Genomic Alterations in Advanced NSCLC

Next-generation sequencing (NGS) of plasma cell-free circulating tumor DNA (cfDNA) enables noninvasive comprehensive genomic analysis. The ability of cfDNA NGS to detect actionable genomic biomarkers in advanced non-small cell lung cancer (NSCLC) has been reported, but there are limited data on real-world usage. Here we prospectively collected the real-world cfDNA NGS data of treatment naive patients and also patients after progression to chemotherapy and/or immunotherapy and tyrosine kinase inhibitors (TKI) with negative tissue results for conventional tesing which we easily encounter in clinical practice. We analyzed data from advanced NSCLC patients who underwent cfDNA NGS (Guardant360; Guardant Health, Inc.) between December 2018 and January 2020 in the clinical practice. Information regarding patient and disease characteristics, treatment decisions, and clinical outcomes were collected. Genomic alterations were considered actionable if they were included in the Onco-KB precision oncology knowledge database and classified in one of four levels of actionability based on the preclinical or clinical evidence. Among 405 patients, 35.1% were female; 81.2% had adenocarcinoma (LuAd). At the time of plasma collection for NGS, 143 patients (35.3 %) were treatment-naïve, 177 patients (43.7%) had progressed after chemotherapy and/or immunotherapy, and 61 patients (15.1%) had progressed after tyrosine kinase inhibitor (TKI). Tumor DNA was detectable in 356 plasma samples (87.9 %). Regarding LuAd potentially actionable level 1-4 genomic alterations were detected in 177 cases (53.6%), of which 82 patients (24.9%) had level 1-2 alterations. Twelve patients who had started treatment based on tissue data had their treatment changed due to cfDNA NGS results. At the time of data cut-off, one of these patients had a partial response, 6 had stable disease, and 5 were not yet evaluated. Real-world cfDNA testing identified actionable genomic alterations in NSCLC not only when tissues were unavailable but also when conventional testing failed to detect actionable biomarkers. This study demonstrated that gene profiling of NSCLC using comprehensive cfDNA NGS can be a more efficient and faster strategy for deciding the therapeutic options at initial diagnosis and after progression.