scholarly journals Influence of preanalytical variables on the quality of cell-free DNA. Biobanking of cell-free DNA material

2022 ◽  
Vol 20 (8) ◽  
pp. 3114
Author(s):  
V. A. Kondratskaya ◽  
M. S. Pokrovskaya ◽  
Yu. V. Doludin ◽  
A. L. Borisova ◽  
A. S. Limonova ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Molecular Mechanisms ◽  
Residual Disease ◽  
Biological Fluids ◽  
Genetic Research ◽  
Pathological Process ◽  
Response To Treatment ◽  
Diagnostic Systems ◽  
Cell Free Dna ◽  
Free Dna

The search for early disease markers and the development of diagnostic systems has recently been expanding within genomics. Genomic deoxyribonucleic acid (DNA), cell-free DNA (cfDNA) and microbiome DNA obtained from different types of samples (tissues, blood and its derivatives, feces, etc.) are used as objects of genetic research. It has been shown that cfDNA that enters the bloodstream, in particular, as a result of apoptosis, necrosis, active tumor secretion and metastasis, is of great importance for studying molecular mechanisms of the pathological process and application in clinical practice. Circulating nucleic acid analysis can be used to monitor response to treatment, assess drug resistance, and quantify minimal residual disease. The review article reflects the following information about the biomaterial: source of cfDNA, methods of cfDNA isolation, storage and use for the diagnosis of certain diseases. Cell-free DNA can be present in biological fluids such as blood, urine, saliva, synovial and cerebrospinal fluid. In most cases, cfDNA is isolated from blood derivatives (serum and plasma), while it is most correct to use blood plasma for cfDNA isolation. Optimal and economically justifiable is the use of ethylenediaminetetra-acetic acid tubes for taking blood and obtaining plasma with subsequent cfDNA isolation. There is evidence that the optimal shelf life in an ethylenediaminetetra-acetic acid tube from the moment of blood sampling to subsequent isolation is a 2-hour interval. After centrifugation, cfDNA in plasma (or serum) can be stored for a long time at a temperature of -80O C. Storage at -20O C is undesirable, since DNA fragmentation increases.

Use of Circulating Cell-Free DNA to Guide Precision Medicine in Patients with Colorectal Cancer

2021 ◽  
Vol 72 (1) ◽  
pp. 399-413
Author(s):  
Van K. Morris ◽  
John H. Strickler
Keyword(s):  
Colorectal Cancer ◽  
Precision Medicine ◽  
Residual Disease ◽  
Cost Effective ◽  
Patient Specific ◽  
Blood Draw ◽  
Cell Free Dna ◽  
Free Dna ◽  
Metastatic Setting ◽  
Diagnostic Technology

Patient-specific biomarkers form the foundation of precision medicine strategies. To realize the promise of precision medicine in patients with colorectal cancer (CRC), access to cost-effective, convenient, and safe assays is critical. Improvements in diagnostic technology have enabled ultrasensitive and specific assays to identify cell-free DNA (cfDNA) from a routine blood draw. Clinicians are already employing these minimally invasive assays to identify drivers of therapeutic resistance and measure genomic heterogeneity, particularly when tumor tissue is difficult to access or serial sampling is necessary. As cfDNA diagnostic technology continues to improve, more innovative applications are anticipated. In this review, we focus on four clinical applications for cfDNA analysis in the management of CRC: detecting minimal residual disease, monitoring treatment response in the metastatic setting, identifying drivers of treatment sensitivity and resistance, and guiding therapeutic strategies to overcome resistance.

A dual target sequencing solution to assess genomic and epigenomic alterations in cell-free DNA with no sample splitting.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3043-3043
Author(s):  
Grace Q. Zhao ◽  
Yun Bao ◽  
Heng Wang ◽  
Wanping Hu ◽  
John Coller ◽  
...  
Keyword(s):  
Cancer Detection ◽  
Dna Analysis ◽  
Residual Disease ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Assay Sensitivity ◽  
Cell Free Dna ◽  
Target Sequencing ◽  
Free Dna ◽  
Dual Analysis

3043 Background: Assessing the genomic and epigenomic changes on plasma cell-free DNA (cfDNA) using next-generation sequencing (NGS) has become increasingly important for cancer detection and treatment selection guidance. However, two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS), let alone one that analyzes both mutation and methylation information in one assay. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for conducting multi-omic analysis. Thus, an assay that is capable of both genomic and epigenomic analysis would be advantageous for clinical research and future diagnostic assays. Methods: Here, we report the performance of Point-n-SeqTM dual analysis, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both genomic and epigenomic analysis without sample splitting. With custom panels of tens to thousands of markers designed with > 99% first-pass success rate, we conducted both performance validation and multi-center, multi-operator, reproducibility studies. Using spike-in titration of cancer cell-line gDNA with known mutation and methylation profiles, Point-n-Seq assay achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions. Benchmarked with conventional targeted sequencing and methylation sequencing, Point-n-Seq solution also demonstrated improved performance, speed and shortened hands-on time. Results: In a pilot clinical study, a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with > 350 hotspot mutations in 22 genes were used in the dual assay. Using 1ml of plasma from late-stage CRC patients, cancer-specific methylation signals were detected in all samples tested, and oncogenic mutations. In an early-stage cohort (33 stage I/II CRC patient ), comparison of the analysis between tumor-informed, personalized-mutation panels (̃100 private SNVs) for each patient and the tumor-independent CRC methylation panels were conducted. The initial results showed that tumor-independent TMS assay achieved a comparable detection compared to the personalized tumor-informed approach. Moreover, cfDNA size information (fragmentome) is also integrated into the analysis of the same Point-n-Seq workflow to improve the assay sensitivity. Conclusions: Point-n-Seq dual analysis is poised to advance both research and clinical applications of early cancer detection, minimal residual disease (MRD), and monitoring.

scholarly journals Cell-free DNA promotes malignant transformation in non-tumor cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Aline Gomes de Souza ◽  
Victor Alexandre F. Bastos ◽  
Patricia Tieme Fujimura ◽  
Izabella Cristina C. Ferreira ◽  
Letícia Ferro Leal ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Tumor Cells ◽  
Malignant Transformation ◽  
Cancer Progression ◽  
Biological Fluids ◽  
Cellular Migration ◽  
Cell Phenotype ◽  
Cell Free Dna ◽  
Prostate Cell ◽  
Free Dna

AbstractCell-free DNA is present in different biological fluids and when released by tumor cells may contribute to pro-tumor events such as malignant transformation of cells adjacent to the tumor and metastasis. Thus, this study analyzed the effect of tumor cell-free DNA, isolated from the blood of prostate cancer patients, on non-tumor prostate cell lines (RWPE-1 and PNT-2). To achieve this, we performed cell-free DNA quantification and characterization assays, evaluation of gene and miRNA expression profiling focused on cancer progression and EMT, and metabolomics by mass spectrometry and cellular migration. The results showed that tumor-free cell DNA was able to alter the gene expression of MMP9 and CD44, alter the expression profile of nine miRNAs, and increased the tryptophan consumption and cell migration rates in non-tumor cells. Therefore, tumor cell-free DNA was capable of altering the receptor cell phenotype, triggering events related to malignant transformation in these cells, and can thus be considered a potential target for cancer diagnosis and therapy.

scholarly journals Real-Time Molecular Monitoring in Acute Myeloid Leukemia With Circulating Tumor DNA

2020 ◽  
Vol 8 ◽  
Author(s):  
Deepshi Thakral ◽  
Ritu Gupta ◽  
Ranjit Kumar Sahoo ◽  
Pramod Verma ◽  
Indresh Kumar ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Disease Progression ◽  
Real Time ◽  
Plasma Cell ◽  
Myeloid Leukemia ◽  
Clonal Evolution ◽  
Response To Treatment ◽  
Cell Free Dna ◽  
Free Dna ◽  
Acute Myeloid

The clonal evolution of acute myeloid leukemia (AML), an oligoclonal hematological malignancy, is driven by a plethora of cytogenetic abnormalities, gene mutations, abnormal epigenetic patterns, and aberrant gene expressions. These alterations in the leukemic blasts promote clinically diverse manifestations with common characteristics of high relapse and drug resistance. Defining and real-time monitoring of a personalized panel of these predictive genetic biomarkers is rapidly being adapted in clinical setting for diagnostic, prognostic, and therapeutic decision-making in AML. A major challenge remains the frequency of invasive biopsy procedures that can be routinely performed for monitoring of AML disease progression. Moreover, a single-site biopsy is not representative of the tumor heterogeneity as it is spatially and temporally constrained and necessitates the understanding of longitudinal and spatial subclonal dynamics in AML. Hematopoietic cells are a major contributor to plasma cell-free DNA, which also contain leukemia-specific aberrations as the circulating tumor-derived DNA (ctDNA) fraction. Plasma cell-free DNA analysis holds immense potential as a minimally invasive tool for genomic profiling at diagnosis as well as clonal evolution during AML disease progression. With the technological advances and increasing sensitivity for detection of ctDNA, both genetic and epigenetic aberrations can be qualitatively and quantitatively evaluated. However, challenges remain in validating the utility of liquid biopsy tools in clinics, and universal recommendations are still awaited towards reliable diagnostics and prognostics. Here, we provide an overview on the scope of ctDNA analyses for prognosis, assessment of response to treatment and measurable residual disease, prediction of disease relapse, development of acquired resistance and beyond in AML.

scholarly journals Heatrich-BS enables efficient CpG enrichment and highly scalable cell-free DNA methylation profiling

2021 ◽  
Author(s):  
Elsie Cheruba ◽  
Ramya Viswanathan ◽  
Pui Mun Wong ◽  
Howard John Womersley ◽  
Yiting Lau ◽  
...  
Keyword(s):  
Bisulfite Sequencing ◽  
Tumor Burden ◽  
Response To Treatment ◽  
Cell Free Dna ◽  
Patient Response ◽  
Free Dna ◽  
Genome Wide ◽  
Quantitative Monitoring ◽  
Genome Bisulfite Sequencing ◽  
Methylation Profiling

Genome wide analysis of cell-free DNA (cfDNA) methylation profile has been shown to be a promising approach for sensitive and specific multi-cancer detection. However, scaling these assays for clinical translation is impractical due to the high cost of whole genome bisulfite sequencing. We showed that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbored the majority of cancer-specific methylation signature. Here, we report on the simple but effective Heat enrichment of CpG-rich regions for Bisulfite Sequencing (Heatrich-BS) platform that enables focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable high accuracy and sensitivity in tumor burden estimation and quantitative monitoring of colorectal patient response to treatment, at much reduced sequencing requirement. Heatrich-BS holds great potential for highly scalable screening and regular monitoring of cancer using liquid biopsy.

In-depth cell-free DNA sequencing reveals genomic landscape of Hodgkin’s lymphoma and facilitates ultrasensitive residual disease detection

Med ◽  
2021 ◽  
Vol 2 (10) ◽  
pp. 1171-1193.e11 ◽  
Author(s):  
Sophia Sobesky ◽  
Laman Mammadova ◽  
Melita Cirillo ◽  
Esther E.E. Drees ◽  
Julia Mattlener ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Hodgkin’S Lymphoma ◽  
Residual Disease ◽  
Hodgkin's Lymphoma ◽  
Disease Detection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Genomic Landscape

Cell-Free DNA for Minimal Residual Disease Monitoring in Multiple Myeloma Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3423-3423 ◽  
Author(s):  
Lenka Kubiczkova-Besse ◽  
Daniela Drandi ◽  
Lenka Sedlarikova ◽  
Stefania Oliva ◽  
Manuela Gambella ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Residual Disease ◽  
Patient Specific ◽  
Cell Free Dna ◽  
Qrt Pcr ◽  
Free Dna ◽  
Diagnostic Samples ◽  
Igh Rearrangement ◽  
Minimal Residual

Abstract Background Circulating nucleic acids, such as cell-free DNA (cf-DNA), are becoming a promising minimally-invasive diagnostic tool for cancer detection. Recent studies demonstrated that tumor-derived cf-DNA can be used to monitor tumor burden and response to treatment in patients (pts) with solid tumors as well as hematological malignancies (Dawson et al, 2013, Armand et al, 2013). In this study we investigated the clinical utility of cf-DNA in the monitoring of minimal residual disease (MRD) of pts with multiple myeloma (MM) carrying the tumor specific immunoglobulin (IGH) rearrangement. Methods Cf-DNA was extracted from 1 ml of serum sample from 13 MM patients enrolled in Italian CRD/MEL-200 and EMN-02 protocols. The total amount of cf-DNA was estimated by fluorometric measurement (median 560 ng, range 15-5158 ng) and the length of fragments was evaluated by high sensitivity dsDNA chips (Agilent). Patient specific clonal IGH rearrangement was identified at the time of diagnosis from bone marrow (BM) genomic DNA (gDNA) as previously reported (Ladetto et al, 2000). For each patient, MRD in BM and peripheral blood (PB) was estimated by real time quantitative PCR (qPCR) using ASO-specific primers and the quantification was based on serial 10-fold dilution standard curves from plasmid carrying the patient specific IGH rearrangement. The amount of IGH rearrangement in cf-DNA (cf-IGH) was estimated by qPCR and droplet digital PCR (ddPCR) (Bio-Rad) on diagnostic and follow up samples and was expressed as the amount of copies per 1 µg of total cf-DNA. qPCR and ddPCR results were interpreted according to the Euro-MRD guidelines (van der Velden et al, 2003). Results Overall, 54 cf-DNA samples from MM serum (13 diagnostic, 41 follow-up samples) were analyzed for the presence of patient specific IGH rearrangement. The most abundant fraction of cf-DNA was 180-220bp, than 350-400bp and 700-10000bp (in 100%, 85% and 68% of samples respectively), whereas longer fragments more often appeared in follow-up samples. By qPCR, cf-IGH at diagnosis were observed in 11/13 diagnostic samples. Only 3/13 pts were quantifiable (116, 85, 187 copies/1 µg of cfDNA) and 8/13 pts were positive but not quantifiable (PNQ) cf-IGH. By ddPCR, levels of cf-IGH at diagnosis were observed in 9/13 pts. 6/13 pts were quantifiable (246, 195, 96, 88, 184, 25 copies/1µg of cfDNA), and only 3/13 pts were PNQ. In follow-up samples, levels of cf-IGH were undetectable by qRT-PCR; however in 5 samples they were PNQ by ddPCR. Interestingly, in one available relapse sample, cf-IGH reappeared again to quantifiable level (61 copies by qRT-PCR and 190 copies by ddPCR). The levels of cf-IGH are quantifiable in samples with higher amount of tumor specific IGH rearrangements in BM or PB; however, no association was observed between cf-IGH level at diagnosis and disease burden estimated by the PCs infiltration in BM or the monoclonal immunoglobulin concentration in blood/urine. Conclusions These data show the potential utility of cf-IGH monitoring in MM pts. Although by qPCR, cf-IGH were detected in 11/13 pts, they were quantifiable only in 3/13 pts and ddPCR was more precise as it was able to quantify cf-IGH in 6/13 pts. Since cf-IGH copies were quantifiable only in diagnostic samples and in 1 available sample at the relapse, we conclude that higher amounts of serum are necessary to overcome the limitation of assay sensitivity. Potential advantages and predictive value, for monitoring tumor marker in a non-invasive manner, need to be further validated on larger cohort of samples using increased amount of cf-DNA. Work was supported by IGA grants NT12130, NT14575. This work is funded by a Black Swan Research Initiative grant by the International Myeloma Foundation "Dynamics of microRNA and cell-free DNA profiles during multiple myeloma progression“. Disclosures Boccadoro: Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria. Palumbo:Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Array BioPharma: Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Sanofi Aventis: Honoraria.

scholarly journals Cell-Free DNA for the Management of Classical Hodgkin Lymphoma

2021 ◽  
Vol 14 (3) ◽  
pp. 207
Author(s):  
Vincent Camus ◽  
Fabrice Jardin
Keyword(s):  
Hodgkin Lymphoma ◽  
Disease Risk ◽  
Residual Disease ◽  
Turnover Time ◽  
Molecular Response ◽  
Classical Hodgkin Lymphoma ◽  
Evaluation Tool ◽  
Easy Method ◽  
Cell Free Dna ◽  
Free Dna

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.

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