Amount Of cfDNA Research Articles

The utility of cfDNA in TGCT patient management: a systematic review.

Background:Testicular germ cell tumors (TGCTs) are the most common young male malignancy with a steadily rising incidence. Standard clinical practice is radical orchidectomy of suspicious lumps followed by histopathological diagnosis and tumor subtyping. This practice can lead to complications and quality of life issues for the patients. Liquid biopsies, especially cell-free DNA (cfDNA), promised to be true surrogates for tissue biopsies, which are considered dangerous to perform in cases of testicular tumors. In this study, we have performed a systematic review on the potential of cfDNA in TGCT patient management, its potential challenges in translation to clinical application and possible approaches in further research.Materials & Methods:The review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines on EuropePMC and PUBMED electronic databases, with the last update being on October 21, 2021. Due to the high heterogeneity in identified research articles, we have performed an overview of their efficacy.Results:Eight original articles have been identified on cfDNA in TGCT patients published from 2004 to 2021, of which six had more than one TGCT patient enrolled and were included in the final analysis. Three studies investigated cfDNA methylation, one has investigated mutations in cfDNA, two have investigated cfDNA amount, and one has investigated cfDNA integrity in TGCT. The sensitivity of cfDNA for TGCT was found to be higher than in serum tumor markers and lower than miR-371a-3p, with comparable specificity. cfDNA methylation analysis has managed to accurately detect teratoma in TGCT patients.Conclusion:Potential challenges in cfDNA application to TGCT patient management were identified. The challenges relating to the biology of TGCT with its low mutational burden and low cfDNA amounts in blood plasma make next-generation sequencing (NGS) methods especially challenging. We have also proposed possible approaches to help find clinical application, including a focus on cfDNA methylation analysis, and potentially solving the challenge of teratoma detection.

Comparison of Methods of Isolation and Quantification of Cell Free DNA from Plasma of Patients with Breast Cancer.

Objective
 Cell-free DNA (cfDNA) released in response to necrosis in cancer patients The objective of this study was to compare the efficiency of two commercial and two manual methods for cell-free DNA extraction. as well as to search for a method that is easy to extract the DNA from the plasma and cost-effective.
 Methods
 Plasma samples seven in number of patients of Breast Cancer was taken. We evaluated DNA quantity and its subsequent amplification obtained by four different cfDNA isolation methods; Modified Phenol Chloroform Isoamyl, Triton Heat Phenol, EpiQUik Circulating Cell-Free DNA isolation Easy kit” (EpiGentek) and “Nucleospin cfDNA kit”. Extracted DNA was quantified using Qubit and quantitative real-time PCR.
 Results
 Quantity of cf DNA varied between different extractions methods of a total of seven samples analyzed. The highest quantity was found from the samples extracted from the Nucleospin XS kit and the extraction efficiency was significantly higher in a pairwise comparison with the other three methods (p-value <0.0001). The concentration of the cfDNA obtained by all four methods was assessed on a Qubit fluorometer. The concentration was higher for the Nucleospin ?MPC?THP?Epiquik kit The qPCR values were consistently higher for Nucleospin XS as compared to all others. This indicates good amplifiability of Nucleospin XS
 Conclusion
 We tested four methods of cf DNA extraction. In our hands, Nucleospin XS gave the best yield and amplifiability. It is a quick and cost-effective method and sensitive for quantification of cfDNA on Real-time PCR. Therefore, it is highly recommended for clinical use of plasma as a liquid biopsy.

Effect of preexamination conditions in a centralized-testing model of non-invasive prenatal screening.

Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF. A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF. We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which issignificantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at-80°C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy. Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.

Non-Invasive Genetic Profiling and Monitoring in Myelodysplastic Syndromes

Introduction. Myelodysplastic syndromes (MDS) are clonal heterogeneous disorders in which molecular studies and cytogenetics are essential for diagnosis, classification and prognosis. Cell-free DNA (cfDNA) analysis has been reported as a reliable non-invasive approach for detecting molecular abnormalities in MDS. However, there is limited information about cytogenetic alterations and monitoring in cfDNA from MDS patients.Objective. To assess and monitor the molecular and cytogenetic abnormalities by next generation sequencing (NGS) using cfDNA from patients with MDS.Patients and methods. Bone marrow (BM) aspirates and peripheral blood (PB) samples were collected from 70 newly diagnosed or treatment-naïve patients with MDS (Table). PB samples from 21 healthy controls were also studied. Molecular characterization was performed in paired samples of BM DNA and cfDNA by NGS in all patients. Libraries were prepared using a custom panel including 48 myeloid-associated genes and genomic regions localized at the most frequently altered chromosomes in MDS (QIAseq Custom DNA Panels, Qiagen) and sequenced using Illumina. 20/70 (28.6%) showed cytogenetic/FISH alterations at diagnosis, 2 of them with infrequent alterations in MDS, not covered by the panel (+14, del(9q)). 6 cases presented chromosome Y loss as a single alteration, not covered by the NGS panel. Copy number variant (CNV) analysis was performed by NGS to detect cytogenetic alterations in both cfDNA and BM, and confirmed by chromosomal microarrays (CMA) in BM DNA (CytoScan/OncoScan, ThermoFisher).Results. We obtained a median amount of cfDNA of 58.4 ng/ml in MDS patients that was significantly higher than that obtained in healthy controls (median 32.4 ng/ml, P=0.023). Sequencing of BM DNA and cfDNA showed a comparable mutational profile (187/201 mutations, 93.0% concordance). The most frequently mutated genes were TET2 (44.3%), SF3B1 (37.1%), ASXL1 (20.0%), DNMT3A (20.0%), SRSF2 (15.7%), ZRSR2 (12.9%) and U2AF1 (12.9%). A strong correlation was observed between the VAFs of BM DNA and cfDNA (r s=0.797, P<0.001). VAFs of SF3B1 mutations detected in cfDNA were significantly higher than VAFs in BM DNA (P=0.016).The analysis of cytogenetic alterations by NGS showed abnormalities in 10/70 MDS patients in both BM DNA and cfDNA. Interestingly, in a patient without analyzable metaphases in karyotype, del(20q) was found by NGS and confirmed by CMA. Overall, CMA and NGS were highly concordant to detect chromosomal aberrations although they do not reach the sensitivity achieved by karyotype/FISH (Figure 1). However, all cytogenetic aberrations detected by NGS in BM DNA were also detected in cfDNA.Molecular and cytogenetic alterations were monitored in sequential samples from 7 cases (median follow up: 13 months, range 10-30). We observed an excellent correlation between the VAF of mutations in BM DNA and ctDNA across multiple matched time points. A decrease in the VAF was detected in patients responding to therapy (either hypomethylating agents or chemotherapy), but not in non-responding patients (Figure 2). Of note, cfDNA analysis also showed cytogenetic evolution in 2 cases not responding to azacitidine (del(12p) and +21). From the two untreated patients, one acquired a subclonal del(7q) not detected by NGS and observed only in few metaphases, and the second patient showed a clonal expansion of the NF1 mutation at the time of AML transformation.Conclusions. Analysis of cfDNA allows the characterization and monitoring of molecular abnormalities in patients with MDS. Cytogenetic alterations were detectable in most cases by NGS in both BM DNA and cfDNA although with a lower detection rate than karyotype/FISH.Acknowledgements. ISCIII-FEDER, PI16/0153, PI19/0005, 2017SGR205, PT20/00023 and XBTC.Figure/Table legends:Table. MDS patients included, classified by disease subtype. (SLD: single lineage dysplasia; MLD: multilineage dysplasia; RS: ring sideroblasts; EB: excess blasts; MDS-U: MDS-unclassifiable)Figure 1. Detection of cytogenetic alterations by conventional karyotype, FISH, CMA and NGS.Figure 2. Monitoring of molecular and cytogenetic alterations in 7 patients with MDS. 5 patients receiving treatment (3 azacitidine, 1 FLAG-IDA + HCT, 1 inhibitor of hypoxia-inducible factor (HIF)) and 2 untreated cases were included. BM VAF dynamics are shown with a dotted line and cfDNA dynamics are shown with a solid line. [Display omitted] DisclosuresBesses: Gilead: Research Funding. Salar: Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Gilead: Research Funding; Celgene: Consultancy, Speakers Bureau. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau.

Interim Analysis of Mmrf Curecloud Research Initiative Identifies High Prevalence and Patterns of Clonal Hematopoiesis of Indeterminate Potential (CHIP) Mutations in a Real World Myeloma Cohort

Background: In order to gain a deeper understanding of the clinical, molecular and immune parameters involved in multiple myeloma (MM) disease initiation, progression and response to treatment, the Multiple Myleoma Research Foundation (MMRF) and its partners launched the CureCloud Research Initiative (NCT03657251), a Direct-to-Patient (DTP) effort aimed at enrolling 5,000 individuals from whom comprehensive molecular and immune analyses are generated from blood specimens and the resulting data aggregated with the correlating clinical information.Methods: To support the molecular characterization of liquid biopsies for CureCloud, a set of myeloma-specific blood-based approaches were developed. The first of such assays is a hybrid selection panel that captures 70 commonly altered MM and Clonal Hematopoiesis of Indeterminate Potential (CHIP) genes detecting somatic variants present in a patient's circulating-free DNA (cfDNA). This MM-70 assay was thoroughly validated to >90% sensitivity for events at 1% variant allele frequency with a specificity of <0.2 FP/Mb when sequenced at 60,000x. A clinical-grade (CLIA) pipeline was established enabling the return of results. Several quality control measures were used to select for CHIP related mutations including filtering out known myeloma driver events, removing high frequency events, and overlapping with previously published CHIP related mutations.Results: 580 subjects were enrolled to the study at abstract submission, including 127 smolderers, 173 newly diagnosed and 265 relapsed participants. 430 cases have been analyzed on the MM-70 assay. A mean cfDNA amount of 31 ng was recovered for input. Mean raw coverage depth of 176,000x was attained, with a mean duplex depth of 1271 (517-2406). 417 cases passed the minimum CLIA validation thresholds. Some of the most frequently mutated genes observed were in the MAPK, DNA repair, epigenetic, cell cycle, and NF-kappaB pathways in agreement with MM retrospective genomic research studies performed on bone marrow aspirates. Of interest, CHIP mutations were detected in a large proportion of cases (174) with their prevalence increasing with age (Figure 1A). 117 of patients had only one CHIP mutation while 57 had two or more with the same genomic loci were recurrently mutated in DNMT3A, TP53, ASXL1, and PPM1D. No significant differences in CHIP were observed between smoldering and overt myeloma subjects (Figure 1B). Interestingly, a greater (>0.1) CHIP Variant Allelic Fraction (VAF) was seen in individuals with higher risk genomic alterations (Figure 1C).Conclusion: This genomic characterization of one of the largest cohorts to date of individuals with myeloma and precursor conditions using a cfDNA-based liquid biopsy assay was able to recapitulate known myeloma-specific mutations and reinforced the importance of CHIP alterations in MM. CHIP mutations are indeed frequent in myeloma, increase with age, and higher burden might be related to disease aggressiveness. [Display omitted] DisclosuresGhobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy.

P–582 High level of concordance between invasive and non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) at day5 and day6–7

Abstract Study question To explore ploidy concordance between invasive and non-invasive PGTA (niPGT-A) at different embryo culture time. Summary answer High level (&amp;gt;84%) of concordance rate for ploidy and sex, sensitivity (&amp;gt;88%), and specificity (76%) were obtained for both day6/7 samples and day5 samples. What is known already The analysis of embryo cell free DNA (cfDNA) that are released into culture media during in vitro embryo development has the potential to evaluate embryo ploidy status. However, obtaining sufficient quality and quantity of cfDNA is essential to achieve interpretable results for niPGT-A. More culture time is expected to be directly proportional to the release of more cfDNA. But embryo culture time is limited due to in-vitro embryo survival potential. Therefore, it is important to estimate the duration of the culture that will provide the maximum cfDNA that can be obtained without adversely affecting the development of the embryo. Study design, size, duration A total of 105 spent culture media (SCM) from day5-day7 blastocyst stage embryos have been included in this cohort study. The cfDNA of SCM samples were amplified and analyzed for niPGT-A by NGS analysis. The SCM samples were divided into 2 subgroups according the embryo culture hours (Day5 and Day6/7 group). The DNA concentration, informativity and euploidy results have then been compared with their corresponding embryos after trophectoderm biopsy (TE) and PGT-A analysis by NGS Participants/materials, setting, methods Embryos cultured until Day3 washed and cultured again in 20µl fresh culture media until embryo biopsy on Day5, 6, or 7. After biopsy SCM samples were immediately collected in PCR tubes and conserved at –20 °C until whole genome amplification by MALBAC® (Yicon Genomics). The TE and SCM samples were analyzed by next-generation sequencing (NGS) using Illumina MiSeq® System. NGS data analysis has been done by Bluefuse Multi Software 4.5 (Illumina) for SCM and TE samples Main results and the role of chance Only the SCM samples which have an embryo with a conclusive result were included in this cohort (n = 105). Overall 97.1% (102/105) of SCM samples gave a successful DNA amplification with a concentration ranging 32.4–128.5ng/µl. Non-informative (NI) results including a chaotic profile (&amp;gt;5 chromosome aneuploidies) were observed in 17 samples, so 83.3%(85/102) of SCM samples were informative for NGS data analysis. Ploidy concordance rate with the corresponding TE biopsies (euploid vs euploid, aneuploid vs aneuploid) was 84.7% (72/85). Sensitivity and specificity were 92,8% and 76,7%, respectively with no significant difference for all parameters for day 6/7 samples compared with day 5 samples. The false-negative rate was 3.5% (3/85), and false-positive rate was 11.7% (10/85). Limitations, reasons for caution The sample size is relatively small. Larger prospective studies are needed. As this is a single-center study, the impact of the variations in embryo culture conditions can be underestimated. Maternal DNA contamination risk cannot be revealed in SCM, therefore the use of molecular markers would increase the reliability. Wider implications of the findings: Non-invasive analysis of embryo cfDNA analyzed in spent culture media demonstrates high concordance with TE biopsy results in both early and late culture time. A non-invasive approach for aneuploidy screening offers important advantages such as avoiding invasive embryo biopsy and decreased cost, potentially increasing accessibility for a wider patient population. Trial registration number Not applicable

The Potential Use of Dynamics Changes of ctDNA and cfDNA in the Perioperative Period to Predict the Recurrence Risk in Early NSCLC.

BackgroundPostoperative circulation tumor DNA (ctDNA) is a promising method to predict the risk of recurrence. However, the amount of ctDNA in patients with early NSCLC is too small. Cell damages caused during the intraoperative period leads to a significant increase in cell free DNA (cfDNA). Whether cfDNA content is restored to the preoperative level within a short time after surgery may indicate the degree of surgical trauma. In this study, dynamic changes of cfDNA combined with ctDNA in the perioperative period of NSCLC were used to explore the possibility of them as a biomarker to indicate the risk of recurrence.MethodsNSCLC patients who planned to undergo radical resection were investigated. 10ml of peripheral blood was collected before, during and 7 days after surgery. DNA concentration was measured, and a 23-gene NGS panel was performed to detect gene mutations. All the patients would be followed-up for at least 18 months.ResultsA total of 7 patients were sampled. The amount of cfDNA before surgery was 36.6 ± 14.7ng, and increased to 127.2 ± 52.2ng during surgery. 7 days after surgery, it dropped to 45.23 ± 9.41ng in 3 patients and rose to 173.7 ± 80.80ng in the remaining 4. Only 1 patient was ctDNA positive after surgery, with decreasing cfDNA, and he was the only one that relapsed and died within 18 months.ConclusionThe use of ctDNA to predict the risk of postoperative recurrence of NSCLC is a very valuable method, and it may be more reliable if combined with the dynamic changes of cfDNA. The amounts of cfDNA are raised by the operation, but will be polarized after surgery in 7 days. Postoperative NSCLC patients with positive ctDNA and reduced cfDNA have a higher risk of recurrence.

Applicability of liquid biopsies to represent the mutational profile of tumor tissue from different cancer entities

Genetic investigation of tumor heterogeneity and clonal evolution in solid cancers could be assisted by the analysis of liquid biopsies. However, tumors of various entities might release different quantities of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) into the bloodstream, potentially limiting the diagnostic potential of liquid biopsy in distinct tumor histologies. Patients with advanced colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), and melanoma (MEL) were enrolled in the study, representing tumors with different metastatic patterns. Mutation profiles of cfDNA, CTCs, and tumor tissue were assessed by panel sequencing, targeting 327 cancer-related genes. In total, 30 tissue, 18 cfDNA, and 7 CTC samples from 18 patients were sequenced. Best concordance between the mutation profile of tissue and cfDNA was achieved in CRC and MEL, possibly due to the remarkable heterogeneity of HNSCC (63%, 55% and 11%, respectively). Concordance especially depended on the amount of cfDNA used for library preparation. While 21 of 27 (78%) tissue mutations were retrieved in high-input cfDNA samples (30–100 ng, N = 8), only 4 of 65 (6%) could be detected in low-input samples (<30 ng, N = 10). CTCs were detected in 13 of 18 patients (72%). However, downstream analysis was limited by poor DNA quality, allowing targeted sequencing of only seven CTC samples isolated from four patients. Only one CTC sample reflected the mutation profile of the respective tumor. Private mutations, which were detected in CTCs but not in tissue, suggested the presence of rare subclones. Our pilot study demonstrated superiority of cfDNA- compared to CTC-based mutation profiling. It was further shown that CTCs may serve as additional means to detect rare subclones possibly involved in treatment resistance. Both findings require validation in a larger patient cohort.

Abstract 572: The liquid biopsy Guardant360 CDx has robust performance at low inputs allowing for high rate of returning patient results

Abstract Background Liquid biopsy tests capable of tumor profiling with NGS have recently received FDA approval for companion diagnostic treatment selection and tumor profiling, demonstrating the clinical validity of the identification of druggable variants with such information. As the amount of cfDNA per volume of plasma varies greatly from patient to patient, and whole blood/plasma volumes from advanced cancer patients are often limited, it would be advantageous for a liquid biopsy to be able to operate with minimal input, providing robust results across a broad spectrum of blood sample volumes and resultant cfDNA input. In this study, we evaluate the performance of Guardant360 CDx with as little as 3 mL whole blood and 5 ng cfDNA input. Methods The primary concern with low input like 3 mL whole blood is excess preservative in a blood collection tube (BCT) relative to whole blood. In a simulated short draw experiment, the same advanced cancer patient provided whole blood in BCTs with standard, elevated (2X), and high (3.3X) preservatives. We evaluated the molecule coverage across the panel at key exons and positions of interest, across genes evaluated for copy number amplifications, and at frequent fusion gene breakpoint positions. Precision and limit of detection (LoD) have been demonstrated at 5 ng input (P200010B) the lowest allowable input. For clinical utility of a liquid biopsy with low cfDNA input, we also assessed the cfDNA input of 87,305 samples tested with Guardant360 CDx or LDT from a single 10mL BCT. Results and Discussion Differences in molecule coverage between the high preservative and standard preservative were found to fall within expected technical variation (the median of log10 differences in exon-level DNA molecule coverage between short draw and reference conditions fell within +/- 0.125), indicating no meaningful loss of sensitivity for detecting somatic variants. Among the 87,305 clinical samples tested by Guardant360 CDx or LDT, median cfDNA yield was 25ng, with 98.5% yielding at least 5 ng, 88.4% at least 10ng, 73.5% at least 15 ng, and 17.9% at least 60ng. Clinically actionable biomarkers are detectable in low input samples. Of the Guardant360 CDx samples yielding 5-15ng cfDNA input, an average of 0.64 clinically significant variants was reported per sample. With the robust analytical performance demonstrated at low inputs, Guardant360 CDx (a 5ng cutoff) may provide tumor profiling results to guide therapy decisions for about 34% more patients compared to some liquid biopsies that use 2 BCTs for minimal 30ng cfDNA yield (a 15 ng cutoff). Retrospective studies for pharma research are often performed with as little as 2mL of plasma, meaning Guardant360 CDx can return results on 88.4% of such samples whereas other technologies may only be able to address 17.9% (extrapolated from the cfDNA yield distribution for 10mL whole blood yielding 4mL plasma). Citation Format: Aaron O. Richardson, Aaron Hardin, Soni Shukla, Jing Zhao, Catalin Barbacioru, Hilda Beas, Carin Espenschied, Adrian Bubie, Jamie Hutchins, Han-Yu Chuang, Tingting Jiang, Darya Chudova. The liquid biopsy Guardant360 CDx has robust performance at low inputs allowing for high rate of returning patient results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 572.

Abstract 575: Enhancing cell-free DNA mutation detection with novel liquid-phase extraction

Abstract Background: Low yields of extracted cell-free DNA (cfDNA) from plasma limit further development of liquid biopsies for early-stage cancer diagnostics and cancer screening applications. Conventional nucleic acid extraction methods have limitations in capturing low levels of cfDNA and risk capturing high levels of high molecular weight contaminating genomic DNA. We developed a liquid-phase based DNA extraction method (PHASIFY™ ENRICH; Phase Scientific International Ltd., Kwun Tong, Hong Kong) that leverages aqueous two-phase systems (ATPS) to purify, concentrate, and enrich for cfDNA to improve mutant signal detection. Methods: The PHASIFY ATPS consists of unique formulations that are optimized to drive cfDNA into one liquid phase and plasma contaminants into the other, achieving simultaneous target isolation and concentration. In a previous study using the PHASIFY technique, we demonstrated improved cfDNA and mutant copy yield over solid-phase methods. We further developed an additional size-selection feature (PHASIFY ENRICH) to remove high molecular weight genomic DNA (&amp;gt;500 bp) and to reduce signal noise for molecular diagnostics. We compared cfDNA recovery and mutation detection from plasma samples extracted with the industry standard QIAamp Circulating Nucleic Acid kit (QCNA; Qiagen, Hilden, Germany) and PHASIFY ENRICH cfDNA extraction kit. According to the respective kit protocols, the cfDNA was extracted from plasma from patients with advanced cancers, including those known to shed low amounts of cfDNA into circulation (e.g., low grade serious ovarian, appendiceal, thyroid, and pancreatic cancer), with known tissue mutation status. Total DNA recovery was determined with the Quant-iT PicoGreen dsDNA Assay Kit. Mutation status was determined with probe-based Droplet Digital PCR (ddPCR; Bio-Rad, Hercules, CA). In a follow up study, we tested a similar cohort of plasma samples that were previously tested negative for cfDNA mutation status when using QCNA cfDNA extraction, despite known positive mutation status from corresponding tumor tissue genotyping. Results: In the head-to-head study against QCNA, 57 plasma samples from 23 unique patients with diverse advanced cancers with known tissue mutation status were used for cfDNA extraction. The PHASIFY ENRICH method resulted in a 153% increase in median mutant copy recovery compared to QCNA (P = 0.01 Wilcoxon Signed-Rank). In the follow up study, 47 plasma samples from 31 patients with diverse advanced cancers and prior cfDNA negative mutation status with tissue positive mutation status were used for extraction. Of the 47samples extracted by PHASIFY ENRICH, 9 samples resulted in a conversion to positive cfDNA mutation status compared to no mutant signals when previously using QCNA. Conclusion: Our results indicate that PHASIFY ENRICH provides improved cfDNA mutation detection by increasing cfDNA recovery and reducing contaminating genomic DNA background. Citation Format: Filip Janku, Helen Huang, David Y. Pereira, Masae Kobayashi, Steven G. Call, Kristen T. Woodbury, Felix Chao, Daniel R. Marshak, Ricky Y. Chiu. Enhancing cell-free DNA mutation detection with novel liquid-phase extraction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 575.

Abstract 569: Correlation of variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors

Abstract Introduction: Two metrics are commonly used to quantify tumor-specific variants in cell-free DNA (cfDNA): the variant allele frequency (VAF), defined as the ratio of the number of variant alleles to wild-type alleles, and mean tumor molecules (MTM)/mL of plasma. The main difference between the two metrics is that molecular disease levels expressed in MTMs account for the total amount of cfDNA. Changes in cfDNA levels, which occur in response to cytotoxic therapies, surgery, and inflammation, have shown to impact the detection and quantification of tumor variants in plasma, leading to misinterpretation of the test results. Here we sought to explore the relationship between VAF and MTM and evaluate the performance of each metric in monitoring molecular residual disease. Methods: We have analyzed 6569 plasma samples from 3389 patients including patients with primary diagnosis of colorectal cancer (n = 2413), breast (n = 170), pancreatic (n = 132), esophageal (n = 86), lung (n = 60), liver (n = 54), other cancers (n = 474) across multiple settings. ctDNA was quantified using a personalized and tumor-informed (bespoke mPCR NGS) assay. We examined the correlation between MTM and VAF for all ctDNA positive plasma samples (n = 1970) from 1283 patients using log-log regression. For patients with longitudinal plasma time points, further analyses on dynamic changes in MTM and VAF was performed to assess the sample-level discrepancy between the two metrics. Furthermore, the absolute values and the dynamic changes in MTM and VAF levels were plotted and compared against clinical truth based on imaging results and patient outcomes. Results: Across all samples median cfDNA concentrations were 7.9 ng/mL (range: 1.3-381.6 ng/mL). Analysis of all ctDNA positive samples revealed a positive correlation between VAF and MTM, with R2 = 0.91 (slope: 0.86). From 3180 longitudinal plasma samples, we found 2.4% (76/3180) of samples, where changes relative to the previous time point were discordant between MTM and VAF. Interestingly, cfDNA concentrations in these samples were significantly altered. In general, cfDNA levels are representative of intrinsic cell turnover. While VAF is directly related to ctDNA, it is inversely related to cfDNA levels. Increased cfDNA levels were seen in the setting of active cytotoxic therapy, perioperatively, and in the setting of advanced disease, suggesting these settings may be prone for VAF to underestimate the true ctDNA burden. Here we provide evidence that failure of VAF to account for the dynamic changes in cfDNA concentration in plasma may result in misinterpretation of the patient's disease burden. This suggests that in certain indications MTM would be more clinically relevant than VAF. Conclusion: Although both VAF and MTM are highly correlated, our analysis shows when cfDNA levels are significantly altered, MTM/mL provides a more accurate measure of molecular disease burden. Citation Format: Antony Tin, Vasily Aushev, Ekaterina Kalashnikova, Raheleh Salari, Svetalana Shchegrova, Gordon Fehringer, Meenakshi Malhotra, Harini Ravi, Himanshu Sethi, Maxim Brevnov, Bernhard Zimmermann, Angel Rodriguez, Paul R. Billings, Alexey Aleshin. Correlation of variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 569.

Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients

Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1–20.3), and intermediate precision ≤ 12.1% (95% CI 9.2–17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.

Personalized Circulating Tumor DNA Dynamically Predicts Response and/or Relapse in Hematological Malignancies

Background: With the recent advent of molecular techniques such as droplet digital PCR (ddPCR), tumor specific genomic component in the plasma or serum, known as circulating tumor DNA (ctDNA), represents one of the most sensitive and noninvasive biomarkers in solid tumors. In the field of hematological malignancies, it remains to be elucidated whether ctDNA monitoring enables accurate prediction of clinical relapse in individual patients. To address this issue, we retrospectively investigated 17 patients with hematological malignancies who had achieved complete or partial remission after first-line induction therapy.Methods: Our cohort consisted of 8 patients with acute myeloid leukemia, 3 with multiple myeloma (MM), 2 with B cell non-Hodgkin's lymphoma, 2 with myelodysplastic syndromes, 1 with B cell acute lymphoblastic leukemia, and 1 with T cell non-Hodgkin's lymphoma. Serial biopsy and matched serum samples were collected at the time of diagnosis (termed “baseline”) and then throughout the treatment course. Cell-free DNA (cfDNA) was extracted from serum samples. We subjected DNA extracted from baseline tumor DNA and buccal swab to next generation sequencing (NGS), identifying one or two somatic driver mutations. We designed personalized ddPCR assays for each somatic mutation identified (n=16). Statistical analysis was performed using the Kaplan-Meier method and the log-rank test. The first day of 1st line induction therapy was set to day 0 for estimation of the disease-free survival (DFS). For subset analysis of patients who relapsed during follow-up, the day of re-induction therapy initiation for relapse is re-set to day 0. Thereafter, the cumulative incidence of relapse, as assessed by ctDNA or by clinical parameters, was estimated and compared.Results: The amount of cfDNA at diagnosis was significantly higher compared with paired cfDNA at remission (p=0.037). NGS screening identified somatic driver mutations in all cases, with 2 mutations found in 2 individuals. Overall, we could construct ddPCR assay for all patients; these mutations included NRAS, KRAS, TP53, GATA2, STAG2, JAK3, MYD88, B2M, NPM1, DNMT3A, U2AF1, and SF3B1 . NGS and ddPCR assays had a good concordance in assessing the same tumor rich samples with regard to the variant allele frequency (VAF) (r2=0.96; p< 0.01). CtDNA could be detected in matched cfDNA from baseline serum. Moreover, the dynamic longitudinal changes of ctDNA VAF reflected the observed overall clinical response, as determined by clinical biomarker or imaging. 8 patients clinically relapsed at a median of 297 days post 1st line therapy (termed “relapsed group”), with the remaining 9 patients disease free at a median of 332 days post 1st line therapy (termed “remission group”). Notably, all of the patients in relapsed group became ctDNA detectable or had ctDNA increased at two consecutive points (termed “ctDNA-increase”). In marked contrast, all of the patients in remission group remained ctDNA undetectable or stable (termed “ctDNA-stable”). Patients with ctDNA-increased had a significantly inferior DFS: 2-year DFS 12.5% in patients with ctDNA-increased vs. 100% in ctDNA-stable, p=0.0115 (Figure 1A). Finally, we compared the timing of molecular relapse and clinical relapse, as assessed by ctDNA and by conventional biomarkers (flowcytometry in leukemia or serum free light chain in MM), respectively, in relapsed group. As a result, molecular relapse as assessed by ctDNA had a median of 30 days lead time (range, 4 to 119 days) over clinical relapse as assessed by conventional clinical parameters (Figure 1B).Conclusions: In summary, personalized ctDNA monitoring could identify patients with impending relapse in advance of established clinical parameters in hematological malignancies. This might allow for clinicians to initiating earlier therapeutic interventions for impending relapse. Further prospective studies in a much larger cohort of hematological malignancies are needed to confirm these findings. [Display omitted] DisclosuresNagamura-Inoue:Tsubakimoto Chain CO.: Research Funding; Rohto Pharmaceutical Co., Ltd.: Research Funding; AMED grant: Research Funding. Tojo:AMED grant: Research Funding.

Personalized Circulating Tumor DNA Predicts Relapse after Allogeneic Hematopoietic Stem Cell Transplantation in Acute Myeloid Leukemia and Myelodysplastic Syndrome

Background: Relapse is a main event of therapy failure for patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) undergoing allogeneic hematopoietic stem cell transplantation (alloSCT). Hence, a novel strategy to detect minimal residual disease (MRD) is required for earlier therapeutic intervention in impending post alloSCT relapse. Recent evidence indicated that screening of driver mutations by next generation sequencing (NGS) followed by detection of circulating tumor DNA (ctDNA) via droplet digital PCR (ddPCR) is a promising approach for MRD monitoring in patients with MDS (Paul Y, et al. Blood. 2017). Here, in this study, we investigated whether serial ctDNA monitoring could identify patients who are most likely to relapse after alloSCT.Methods: We performed a retrospective study in 31 patients with AML and MDS undergoing alloSCT. Cell-free DNA (cfDNA) was extracted from serum samples. We subjected primary tumor DNA, extracted from a diagnostic peripheral blood or bone marrow (BM), and buccal swab DNA, to NGS, identifying one or two somatic driver mutations. We designed unique ddPCR assays for each somatic mutation identified (n=25). The primary endpoint was cumulative incidence of relapse rate (CIR) and the secondary endpoint was overall survival (OS). CIR and OS were calculated by the Kaplan-Meier method. The log-rank test was used for group comparison.Results: 31 patients were enrolled in our study: our cohort consisted of 10 patients with de novo AML, 15 with AML with myelodysplasia related changes, and 6 with MDS. The median age was 52 years (17-68 years) and 5 patients (16%) are in complete remission at alloSCT. Stem cell source included related bone marrow (n=2) and cord blood (n=29). The preparative regimen included myeloablative conditioning (n=17) and reduced intensity conditioning (n=14). NGS screening identified 26 somatic driver mutations in 30 of 31 (97%) cases, with 2 mutations found in 5 individuals. Overall, we could construct ddPCR assays for 29 of 31 (94%) patients: these mutations included NRAS, TP53, NPM1, DNMT3A, GATA2, etc. NGS and ddPCR assays had a good concordance in assessing the same tumor rich samples with regard to the variant allele frequency (VAF).The amount of cfDNA at diagnosis was significantly higher compared with paired cfDNA at remission (n=23; p=0.0133, paired t-test). Of 37 matched serial time points, VAF between BM and serum ctDNA indicates apparent correlation (r2=0.92, p<0.0001). CtDNA was detected in 10 of 25 (40%) samples at 1 month after alloSCT and 7 of 21 (33%) samples at 3 months (termed “ctDNA1-positive” and “ctDNA3-positive”, respectively).We next evaluated whether the positivity of ctDNA at 1 month and 3 months after alloSCT was associated with higher CIR and lower OS. As expected, patients with ctDNA1-positive and ctDNA3-positive had significantly superior CIR and inferior OS: 2-year CIR 51% in ctDNA1-positive patients vs. 9% in negative patients, p=0.0005 (Figure 1A); 2-year OS 48% in ctDNA1-positive patients vs. 83% in negative patients, p=0.0152 (Figure 1B); 2-year CIR 57% in ctDNA3-positive patients vs. 23% in negative patients, p=0.0055 (Figure 1C); 2-year OS 50% in ctDNA3-positive patients vs. 88% in negative patients, p=0.0344 (Figure 1D), respectively.Conclusions: In summary, personalized ctDNA monitoring could detect MRD, which alloSCT has failed to eradicate, and thus identify patients with high-risk of relapse. This might allow for clinicians to initiating earlier therapeutic interventions for impending relapse. Further prospective studies in a much larger cohort of AML and MDS undergoing alloSCT are required to confirm these findings.Figure 1. CIR and OS based on ctDNA status at 1 month and 3 months after alloSCT [Display omitted] DisclosuresNagamura-Inoue:Tsubakimoto Chain CO.: Research Funding; AMED grant: Research Funding; Rohto Pharmaceutical Co., Ltd.: Research Funding. Tojo:AMED grant: Research Funding.

Molecular Characterization of Waldenström's Macroglobulinemia Patients Using Peripheral Blood Cell-Free Tumor DNA

BackgroundMutational characterization of Waldenström's macroglobulinemia (WM) currently relies on DNA from CD19-selected cells derived from bone marrow (BM) aspirates, which is an invasive technique, associated with significant patient discomfort. Peripheral blood cell-free tumour DNA (cf or ctDNA) has been recently shown to be a powerful non-invasive molecular biomarker in monitoring tumor status in several cancers. In this study, we aimed to characterize the mutational status of WM/IgM MGUS patients by using peripheral blood plasma-derived cfDNA and using matched tumor DNA (tDNA) from BM-CD19+ selected cells as a control, in order to determine whether peripheral blood , by means of cfDNA, can be used as an additional diagnostic tool in identifying the mutational profile of WM.MethodsA total of 30 consecutive patients with IgM monoclonal gammopathies were included in this study. All patients gave a written informed consent for sample collection and analysis. Peripheral blood (10-12mL) was collected in EDTA tubes and DNA was extracted using the MagMax cell free DNA isolation kit (ThermoFisher Scientific). cfDNA concentration was measured using the Qubit fluorometer. The quantities of cfDNA varied between 1.5 ng/μL and 8 ng/μL. BM aspirates were collected at the same time with peripheral blood, and were processed for CD19 enrichment, using CD19 magnetic beads (Miltenyi Biotech), followed by DNA extraction (Invitrogen). The paired tDNA and cfDNA samples from the 30 patients were analyzed for the MYD88 L265P and for CXCR4 mutations. The presence of L265P mutation was initially assessed by Allele-Specific PCR (AS-PCR) and then confirmed with direct sequencing. The presence of CXCR4 mutations was assessed with direct sequencing.ResultsThe study included patients with symptomatic WM before initiation of any therapy (N=4) , with WM under therapy (N=4) or after completion of therapy (N=12), with asymptomatic WM (N=8) and with IgM MGUS (N=2). The median age of the patients was 70 years (range 22-87). The median bone marrow infiltration of the tested samples was 28% (range 5 to 90%) and median IgM levels were 1510 mg/dL (range 80 to 7310 mg/dL).In all, but one patient, (29/30) the L265P mutation was detected in both cfDNA and tDNA (100% concordance). This mutation was initially detected by AS-PCR which was validated by direct sequencing in all patients. Assessment of CXCR4 mutation status was feasible in 29 patients; in one patient CXCR4 sequencing was unsuccessful in both cfDNA and tDNA. Mutations in CXCR4 were detected in 24% (7/29) of patients. The pathogenic mutation S338X (rs104893626) was present in 2 (7%) patients and was identified in both cfDNA and tDNA. In one patient the L50X (1/29) truncating mutation was found in tDNA but not in cfDNA. Two patients harbored the F29L (2/29) mutation and another two (2/29) the P31T mutation, both in cfDNA and in tDNA; these two mutations have not been previously reported. Synonymous mutations were also found in 10% of the tested patients in both cfDNA and tDNA. Thus, the concordance for CXCR4 mutations identified by direct sequencing of cfDNA and tDNA was 97% (in 28/29 patients).ConclusionsOur study demonstrates that in patients with IgM monoclonal gammopathies peripheral blood cell-free DNA can be used as a non-invasive method to detect the MYD88 L265P mutation with excellent concordance to BM CD19+ derived tumor DNA. Furthermore, CXCR4 mutations could also be detected with high concordance to tDNA by direct sequencing. This method could be more cost effective, as it avoids the expenses and time needed for CD19 selection, either BM or peripheral blood derived. Peripheral blood cfDNA could be used as an initial test to assess the presence of MYD88 L265P and CXCR4 mutations, so that BM testing can be limited to patients with indeterminate results. Updated results will be presented in the meeting. Further investigation is ongoing in order to assess tumor dynamics using peripheral blood. DisclosuresTerpos:Janssen: Honoraria, Research Funding; Genesis/Celgene: Honoraria, Other: DMC member, Research Funding; Abbvie: Honoraria; Takeda: Honoraria, Other: SC member; Amgen: Honoraria, Other: SC member, Research Funding; GSK: Honoraria; BMS: Honoraria. Kastritis:Prothena: Honoraria; Janssen: Honoraria; Genesis pharma: Honoraria; Takeda: Honoraria; Amgen: Honoraria, Research Funding. Dimopoulos:Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology: Consultancy, Honoraria, Other: Advisory Committee: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology; Novartis: Consultancy, Honoraria; Genesis Pharma: Research Funding.

Feasibility of Cell-Free DNA Collection and Clonal Immunoglobulin Sequencing in South African Patients With HIV-Associated Lymphoma.

PURPOSEDiagnosis of AIDS lymphoma in low-resource settings, like South Africa, is often delayed, leaving patients with limited treatment options. In tuberculosis (TB) endemic regions, overlapping signs and symptoms often lead to diagnostic delays. Assessment of plasma cell-free DNA (cfDNA) by next-generation sequencing (NGS) may expedite the diagnosis of lymphoma but requires high-quality cfDNA.METHODSPeople living with HIV with newly diagnosed aggressive B-cell lymphoma and those with newly diagnosed TB seeking care at Chris Hani Baragwanath Academic Hospital and its surrounding clinics, in Soweto, South Africa, were enrolled in this study. Each participant provided a whole blood specimen collected in cell-stabilizing tubes. Quantity and quality of plasma cfDNA were assessed. NGS of the immunoglobulin heavy chain was performed.RESULTSNine HIV+ patients with untreated lymphoma and eight HIV+ patients with TB, but without lymphoma, were enrolled. All cfDNA quantity and quality metrics were similar between the two groups, except that cfDNA accounted for a larger fraction of recovered plasma DNA in patients with lymphoma. The concentration of cfDNA in plasma also trended higher in patients with lymphoma. NGS of immunoglobulin heavy chain showed robust amplification of DNA, with large amplicons (> 250 bp) being more readily detected in patients with lymphoma. Clonal sequences were detected in five of nine patients with lymphoma, and none of the patients with TB.CONCLUSIONThis proof-of-principle study demonstrates that whole blood collected for cfDNA in a low-resource setting is suitable for sophisticated sequencing analyses, including clonal immunoglobulin NGS. The detection of clonal sequences in more than half of patients with lymphoma shows promise as a diagnostic marker that may be explored in future studies.

Using cfRNA as a tool to evaluate clinical treatment outcomes in patients with metastatic lung cancers and other tumors.

Aim: We report an exploratory analysis of cfRNA as a biomarker to monitor clinical responses in non-small cell lung cancer (NSCLC), breast cancer, and colorectal cancer (CRC). An analysis of cfRNA as a method for measuring PD-L1 expression with comparison to clinical responses was also performed in the NSCLC cohort. Methods: Blood samples were collected from 127 patients with metastatic disease that were undergoing therapy, 52 with NSCLC, 50 with breast cancer, and 25 with CRC. cfRNA was purified from fractionated plasma, and following reverse transcription (RT), total cfRNA and gene expression of PD-L1were analyzed by real-time polymerase chain reaction (qPCR) using beta-actin expression as a surrogate for relative amounts of cfDNA and cfRNA. For the concordance study of liquid biopsies and tissue biopsies, the isolated RNA was analyzed by RNAseq for the expressions of 13 genes. We had to close the study early due to a lack of follow-up during the Covid-19 pandemic. Results: We collected a total of 373 blood samples. Mean cfRNA PCR signals after RT were about 50-fold higher than those of cfDNA. cfRNA was detected in all patients, while cfDNA was detected in 88% of them. A high concordance was found for the expression levels of 13 genes between blood and solid tumor tissue. Changes in cfRNA levels followed over the course of treatments were associated with response to therapy, increasing in progressive disease (PD) and falling when a partial response (PR) occurred. The expression of PD-L1 over time in patients treated with immunotherapy decreased with PR but increased with PD. Pre-treatment levels of PD-L1 were predictive of response in patients treated with immunotherapy. Conclusion: Changes in cfRNA correlate with clinical response to the therapy. Total cfRNA may be useful in predicting clinical outcomes. PD-L1 gene expression may provide a biomarker to predict response to PD-L1 inhibition.

Circulating cell-free DNA species affect the risk of hepatocellular carcinoma in treated chronic hepatitis B patients.

Hepatocellular carcinoma (HCC) may still develop in chronic hepatitis B (CHB) patients even under effective long-term oral antiviral therapy, but its pathogenesis in the setting of long-standing inhibition of viral replication has not been completely elucidated. We investigated whether species of circulating cell-free DNA (cfDNA) may be involved in the process of hepatocarcinogenesis in treated CHB patients. Serum samples were obtained from HBeAg-negative CHB patients with (HCC cases, n=37) or without HCC development during the first 5years of oral antiviral therapy (controls, n=74). HCC cases and controls were matched 1:2 for age, sex and platelets. Determination of different circulating cfDNA species (before HCC diagnosis in HCC cases) including total cfDNA quantity, levels of Alu repeat DNA and RNase P coding DNA, copies of mitochondrial DNA and levels of 5-methyl-2'-deoxycytidine as an indicator of DNA methylation was performed. HCC cases compared with controls had higher median levels of Alu247 (123 vs 69 genomic equivalent, p=.042) and RNase P coding DNA (68 vs 15 genomic equivalent, p<.001). In contrast, median cfDNA concentration, Alu115 levels, Alu247/Alu115 ratio as an index of DNA integrity and mitochondrial DNA copies did not differ significantly between HCC cases and controls. Receiver operating characteristic curve analysis showed that levels RNase P coding DNA offered good prediction of subsequent HCC development (c-statistic: 0.80, p<.001). In conclusion, serum levels of RNase P coding DNA are increased years before HCC diagnosis and could be potentially helpful in the prediction of the HCC risk in treated HBeAg-negative CHB patients.

LiBis: an ultrasensitive alignment augmentation for low-input bisulfite sequencing.

The cell-free DNA (cfDNA) methylation profile in liquid biopsy has been utilized to diagnose early-stage disease and estimate therapy response. However, typical clinical procedures are capable of purifying only very small amounts of cfDNA. Whole-genome bisulfite sequencing (WGBS) is the gold standard for measuring DNA methylation; however, WGBS using small amounts of fragmented DNA introduces a critical challenge for data analysis, namely a low-mapping ratio. The resulting low sequencing depth and low coverage of CpG sites genome-wide is a bottleneck for the clinical application of cfDNA-based WGBS assays. We developed LiBis (Low-input Bisulfite Sequencing), a novel method for low-input WGBS data alignment. By dynamically clipping initially unmapped reads and remapping clipped fragments, we judiciously rescued those reads and uniquely aligned them to the genome. By substantially increasing the mapping ratio by up to 88%, LiBis dramatically improved the number of informative CpGs and the precision in quantifying the methylation status of individual CpG sites. LiBis significantly improved the cost efficiency of low-input WGBS experiments by dynamically removing contamination introduced by random priming. The high sensitivity and cost effectiveness afforded by LiBis for low-input samples will allow the discovery of genetic and epigenetic features suitable for downstream analysis and biomarker identification using liquid biopsy.

Diagnostic value of quantification of circulating free DNA for gall bladder cancer using a chemiluminescence DNA biosensor system based on DNA G-quadruplex/ hemin enzyme

Gall bladder cancer (GBC) is an insidious but rapidly progressed disease with a poor prognosis and high mortality rate. To explore a novel method for GBC diagnosis, we quantified circulating free DNA (cfDNA) in serum samples from 228 participants, including 83 patients with GBC, 75 patients with cholecystitis, and 70 healthy donors, using a chemiluminescence DNA biosensor system based on DNA G-quadruplex/hemin enzyme. We measured β-actin gene expression to evaluate serum cfDNA levels representing as chemiluminescence intensity with the addition of sufficient probes. We analyzed associations of cfDNA quantities in serum samples and corresponding pathological stages and found that the concentration of cfDNA was significantly higher in GBC group than in the healthy control and cholecystitis groups. The levels of cfDNA were significantly associated with TNM stage, lymph node involvement, metastasis, and jaundice. The ROC curves showed that the diagnostic value of chemiluminescence DNA biosensor system was nearly equivalent to that of qPCR. Our method can distinguish patients with GBC from healthy donors and patients with cholecystitis clearly; however, this method was not available to distinguish patients with cholecystitis from the healthy controls. In summary, cfDNA maybe serve as a new diagnostic and noninvasive marker for the diagnosis of GBC using chemiluminescence DNA biosensor system based on DNA G-quadruplex/hemin enzyme.