scholarly journals From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3002
Author(s):  
Kendra K. Maass ◽  
Paulina S. Schad ◽  
Agnes M. E. Finster ◽  
Pitithat Puranachot ◽  
Fabian Rosing ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Pcr Amplification ◽  
Genomic Analysis ◽  
Digital Pcr ◽  
Great Promise ◽  
Sample Collection ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Dna And Rna ◽  
Free Dna

Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample.

scholarly journals Rapid response of stage IV colorectal cancer with APC/TP53/KRAS mutations to FOLFIRI and Bevacizumab combination chemotherapy: a case report of use of liquid biopsy

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Alexander Hendricks ◽  
Philip Rosenstiel ◽  
Sebastian Hinz ◽  
Greta Burmeister ◽  
Christoph Röcken ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Treatment Response ◽  
Liquid Biopsy ◽  
Metastatic Cancer ◽  
Stage Iv ◽  
Liquid Biopsies ◽  
Kras Mutations ◽  
Cell Free Dna ◽  
Stage Iv Colorectal Cancer ◽  
Free Dna

Abstract Background Liquid biopsies of blood plasma cell free DNA can be used to monitor treatment response and potentially detect mutations that are present in resistant clones in metastatic cancer patients. Case presentation In our non-interventional liquid biopsy study, a male patient in his fifties diagnosed with stage IV colorectal cancer and polytope liver metastases rapidly progressed after completing chemotherapy and deceased 8 months after diagnosis. Retrospective cell free DNA testing showed that the APC/TP53/KRAS major clone responded quickly after 3 cycles of FOLFIRI + Bevacizumab. Retrospective exome sequencing of pre-chemotherapy and post-chemotherapy tissue samples including metastases confirmed that the APC/TP53/KRAS and other major clonal mutations (GPR50, SLC5A, ZIC3, SF3A1 and others) were present in all samples. After the last chemotherapy cycle, CT imaging, CEA and CA19–9 markers validated the cfDNA findings of treatment response. However, 5 weeks later, the tumour had rapidly progressed. Conclusion As FOLFIRI+Bevacizumab has recently also been associated with sustained complete remission in a APC/TP53/KRAS triple-mutated patient, these driver genes should be tested and monitored in a more in-depth manner in future patients. Patients with metastatic disease should be monitored more closely during and after chemotherapy, ideally using cfDNA.

Liquid biopsies of plasma exosomal nucleic acids, plasma cell-free DNA, and survival of patients with advanced cancers.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. 11551-11551
Author(s):  
Lino Moehrmann ◽  
Helen J. Huang ◽  
David S. Hong ◽  
Apostolia Maria Tsimberidou ◽  
Siqing Fu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Prognostic Factor ◽  
Kras Mutation ◽  
Tumor Tissue ◽  
Digital Pcr ◽  
Mutation Testing ◽  
Clinical Testing ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Free Dna

11551 Background: Blood-based liquid biopsies offer easy accessible genomic material for molecular diagnostics in cancer. Commonly used cell-free DNA (cfDNA) originates from dying cells. In contrast exosomal nucleic acid (exoNA) originates from living cells, which can better reflect underlying cancer biology. Methods: We isolated exoNA (EXO52) and cfDNA (QIAamp Circulating Nucleic Acid kit) from plasma of patients with progressing advanced cancers and tested for BRAFV600, KRASG12/G13, and EGFRexon19del/L858R mutations using next-generation sequencing (EXO1000), droplet digital PCR (ddPCR, QX200) and BEAMing digital PCR. The results were compared to clinical testing of archival tumor tissue and correlated with survival. Results: Of the 43 patients (colorectal cancer, 20; melanoma, 8; non-small cell lung cancer, 6; ovarian cancer, 2; papillary thyroid cancer, 2; other cancers, 5) 41 had a mutation in the tumor tissue (20 [47%] BRAF mutation, 17 [40%] KRAS mutation and 4 [9%] EGFR mutation). Mutation testing of plasma exoNA from all 43 patients detected 39 (95%) of 41 mutations present in tumor tissue with 100% specificity. Mutation testing of plasma cfDNA from 39 patients using ddPCR detected 33 (89%) of 37 mutations present in tumor and testing of plasma cfDNA from 37 patients using BEAMing detected 34 (97%) of 35 mutations present in tumor tissue; however, both cfDNA methods reported an additional KRAS mutation not present in tumor tissue. Patients with high mutation allele frequency (MAF, > median) had shorter median survival compared to patients with low MAF ( < median) when using exoNA (5.9 vs. 11.8 months, P= 0.006), but not cfDNA ddPCR (6.0 vs. 7.4 months, P= 0.06) or cfDNA BEAMing (6.5 vs. 7.4 month, P= 0.07). High MAF in exoNA was an independent prognostic factor for survival in multicovariate analysis (HR 0.13, P= 0.017). Conclusions: Mutation testing of plasma exoNA for common BRAF, KRAS, and EGFR mutations has high sensitivity compared to clinical testing of archival tumor tissue and better specificity than PCR testing of plasma cfDNA. High MAF in exoNA is the independent prognostic factor for shorter survival.

Comparison of circulating tumor cells and cell-free DNA in the molecular characterization of patients with head and neck cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18521-e18521
Author(s):  
Santiago Cabezas-Camarero ◽  
Vanesa García-Barberán ◽  
Virginia De la Orden-García ◽  
Beatriz Mediero-Valeros ◽  
Isabel Díaz-Millán ◽  
...  
Keyword(s):  
Head And Neck Cancer ◽  
Parotid Gland ◽  
Head And Neck ◽  
Molecular Characterization ◽  
Neck Cancer ◽  
Liquid Biopsy ◽  
Digital Pcr ◽  
Cell Free Dna ◽  
Free Dna

e18521 Background: The role of liquid biopsy in diagnosis and therapy monitoring in patients with head and neck cancer has been much less studied compared to other cancers. Our aim was to evaluate the perfomance in the isolation and recovery for molecular characterization of circulating tumour cells (CTC) of a new immunoafinity-based method and to compare it with the molecular diagnostic yield of plasma cell-free DNA. Methods: Patients with recurrent/metastatic (RM) head and neck cancer (HNC) were enrolled prospectively. Forty mililiters (ml) of plasma were collected at one or several time-points. First blood draw was always collected before starting a new therapeutic intervention or at the time of radiologic progression. For CTC detection and isolation, either anti-EpCAM or both anti-EpCAM + anti-EGFR antibodies were used. Digital PCR and castPCR were used to study KRAS and PI3KCA mutations in non-squamous HNC. A 15-gene customized NGS panel was used to characterized both CTC and cfDNA in patients with squamous HNC. Results: Between February 2016 and October 2018, 14 patients with R/M HNC were included (n = 1 local-only disease, n = 10 local and distant disease, n = 3 distant-only disease). Squamous histology (S): n = 9. Non-squamous (NS): n = 5 (1 naso-ethmoidal intestinal-type adenocarcinoma, 1 parotid gland exadenoma pleomorfic carcinoma, 2 parotid-gland salivary duct carcinomas (SDC), 1 parotid-gland high-grade neuroendocrine carcinoma). Twenty-five CTC determinations were performed. In 5 patients serial CTC determinations were performed. Median CTC was 4 (min-max: 0-49). Median CTC among 11 CTC determinations in S-HNC was 4 (min-max: 0-49). Median CTC was 3 CTC (min-max: 0-26) among the 14 determinations performed in NS-HNC. Digital PCR unveiled mutations in CTC and in cfDNA in 2 of 4 patients tested with NS histology (KRAS, PIK3CA), with one of them being concordant for the specific mutation. NGS unveiled mutations in CTC in 7/9 patients and in cfDNA in 6/9 patients, with only one loci-concordant case between CTC and plasma. Conclusions: IsoFlux detected CTC in the majority of patients with R/M HNC, regardless of the histologic type, and allowed for molecular characterization of CTC using different techniques for mutational analysis. Both NGS and digital PCR allowed for the detection in cell-free DNA of commonly mutated genes in HNC. Liquid biopsy should be more actively studied in this disease in order to better define its role in diagnosis and therapeutic monitoring.

scholarly journals Liquid Biopsy Analysis of FGFR3, TERT Promoter and STAG2 Hotspot Mutations for Disease Surveillance in Bladder Cancer

2020 ◽  
pp. 1-7
Author(s):  
Victor Romanov ◽  
Dimitri Gnatenko ◽  
Edward Forsyth ◽  
Liang Xiaohui ◽  
Olga Povcher ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Sensitivity And Specificity ◽  
Liquid Biopsy ◽  
Disease Surveillance ◽  
Digital Pcr ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tert Promoter ◽  
Mutational Status ◽  
Muscle Invasive

Patients with non-muscle invasive bladder cancer (NMIBC) are followed by frequent cystoscopies. Innovative approaches partly replacing cystoscopy (uncomfortable, expensive, low sensitive procedure) are demanded. The current study aims to establish a fast, reliable, non-invasive, and inexpensive procedure for NMIBC patient surveillance. Liquid biopsy is a reliable source of biomarkers for cancer patient monitoring. Urine is the most suitable biological liquid to search for bladder cancer biomarkers. Cell-free DNA in urine represents tumor-related mutations for several cancers, including the bladder. We investigated mutations in FGFR3, TERT promoter, and STAG2 as markers for diagnostics and follow-up in NMIBC. Digital PCR was used to detect mutations in urine-derived cell-free DNA. The sensitivity and specificity of the markers in relation to clinical outcomes served as criteria of the assay efficiency. The sensitivity with a single marker (TERT) reached 87%, with a specificity of 77%. Combining two biomarkers (TERT+FGFR3) increased the specificity of the assay to 100% with a sensitivity of 72%. Different mutational status of STAG2 can indicate NMIBC presence or recurrence. Therefore, applying the suggested combination of biomarkers with simple detection procedures to larger patient cohorts will allow developing procedures for BC detection and surveillance with optimal sensitivity and specificity. Based on the results of this proof-in-concept study, we conclude that this simple, fast and inexpensive assay can add diagnostic and prognostic value to cystoscopy/cytology analysis of NMIBC patients.

scholarly journals The pitfalls and promise of liquid biopsies for diagnosing and treating solid tumors in children: a review

2020 ◽  
Vol 179 (2) ◽  
pp. 191-202 ◽  
Author(s):  
Ruben Van Paemel ◽  
Roos Vlug ◽  
Katleen De Preter ◽  
Nadine Van Roy ◽  
Frank Speleman ◽  
...  
Keyword(s):  
Solid Tumors ◽  
Liquid Biopsy ◽  
Residual Disease ◽  
Therapy Response ◽  
Response Monitoring ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Pediatric Solid Tumors ◽  
Free Dna ◽  
Tumor Types

AbstractCell-free DNA profiling using patient blood is emerging as a non-invasive complementary technique for cancer genomic characterization. Since these liquid biopsies will soon be integrated into clinical trial protocols for pediatric cancer treatment, clinicians should be informed about potential applications and advantages but also weaknesses and potential pitfalls. Small retrospective studies comparing genetic alterations detected in liquid biopsies with tumor biopsies for pediatric solid tumor types are encouraging. Molecular detection of tumor markers in cell-free DNA could be used for earlier therapy response monitoring and residual disease detection as well as enabling detection of pathognomonic and therapeutically relevant genomic alterations.Conclusion: Existing analyses of liquid biopsies from children with solid tumors increasingly suggest a potential relevance for molecular diagnostics, prognostic assessment, and therapeutic decision-making. Gaps remain in the types of tumors studied and value of detection methods applied. Here we review the current stand of liquid biopsy studies for pediatric solid tumors with a dedicated focus on cell-free DNA analysis. There is legitimate hope that integrating fully validated liquid biopsy–based innovations into the standard of care will advance patient monitoring and personalized treatment of children battling solid cancers. What is Known:• Liquid biopsies are finding their way into routine oncological screening, diagnosis, and disease monitoring in adult cancer types fast.• The most widely adopted source for liquid biopsies is blood although other easily accessible body fluids, such as saliva, pleural effusions, urine, or cerebrospinal fluid (CSF) can also serve as sources for liquid biopsies What is New:• Retrospective proof-of-concept studies in small cohorts illustrate that liquid biopsies in pediatric solid tumors yield tremendous potential to be used in diagnostics, for therapy response monitoring and in residual disease detection.• Liquid biopsy diagnostics could tackle some long-standing issues in the pediatric oncology field; they can enable accurate genetic diagnostics in previously unbiopsied tumor types like renal tumors or brain stem tumors leading to better treatment strategies

scholarly journals Cell-Free DNA-Methylation-Based Methods and Applications in Oncology

Biomolecules ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1677
Author(s):  
Francesca Galardi ◽  
Francesca De Luca ◽  
Dario Romagnoli ◽  
Chiara Biagioni ◽  
Erica Moretti ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Liquid Biopsy ◽  
Dynamic Assessment ◽  
Disease Status ◽  
Great Promise ◽  
Tumor Type ◽  
Cell Of Origin ◽  
Cell Free Dna ◽  
Free Dna ◽  
Input Sample

Liquid biopsy based on cell-free DNA (cfDNA) enables non-invasive dynamic assessment of disease status in patients with cancer, both in the early and advanced settings. The analysis of DNA-methylation (DNAm) from cfDNA samples holds great promise due to the intrinsic characteristics of DNAm being more prevalent, pervasive, and cell- and tumor-type specific than genomics, for which established cfDNA assays already exist. Herein, we report on recent advances on experimental strategies for the analysis of DNAm in cfDNA samples. We describe the main steps of DNAm-based analysis workflows, including pre-analytics of cfDNA samples, DNA treatment, assays for DNAm evaluation, and methods for data analysis. We report on protocols, biomolecular techniques, and computational strategies enabling DNAm evaluation in the context of cfDNA analysis, along with practical considerations on input sample requirements and costs. We provide an overview on existing studies exploiting cell-free DNAm biomarkers for the detection and monitoring of cancer in early and advanced settings, for the evaluation of drug resistance, and for the identification of the cell-of-origin of tumors. Finally, we report on DNAm-based tests approved for clinical use and summarize their performance in the context of liquid biopsy.

scholarly journals Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol

Cancers ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 458 ◽  
Author(s):  
Laure Sorber ◽  
Karen Zwaenepoel ◽  
Julie Jacobs ◽  
Koen De Winne ◽  
Sofie Goethals ◽  
...  
Keyword(s):  
High Speed ◽  
Fragment Size ◽  
Molecular Profile ◽  
Great Promise ◽  
Cell Free Dna ◽  
Dna And Rna ◽  
Free Dna ◽  
Droplet Pcr ◽  
One Step ◽  
Temperature Time

The combined analysis of circulating cell-free (tumor) DNA (cfDNA/ctDNA) and circulating cell-free (tumor) RNA (cfRNA/ctRNA) shows great promise in determining the molecular profile of cancer patients. Optimization of the workflow is necessary to achieve consistent and reproducible results. In this study, we compared five centrifugation protocols for the optimal yield of both cfDNA/ctDNA and cfRNA/ctRNA. These protocols varied in centrifugation speed, ambient temperature, time, and number of centrifugation steps. Samples from 33 participants were collected in either BD Vacutainer K2EDTA (EDTA) tubes or cell-free DNA BCT® (Streck) tubes. cfDNA concentration and fragment size, and cfRNA concentration were quantitated in all samples by digital droplet PCR (ddPCR) and quantitative PCR (qPCR). The KRAS-mutated ctDNA and ctRNA fraction was determined via ddPCR. In EDTA tubes, the protocol generating both plasma and platelets was found to produce high quality cfDNA and cfRNA concentrations. Two-step, high-speed centrifugation protocols were associated with high cfDNA but low cfRNA concentrations. High cfRNA concentrations were generated by a one-step, low-speed protocol. However, this coincided with a high amount of genomic DNA (gDNA) contamination. In Streck tubes, two-step, high-speed centrifugation protocols also generated good quality, high cfDNA concentration. However, these tubes are not compatible with cfRNA analysis.

Development and analytical validation of a 523-gene clinical assay for cell-free DNA.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 3039-3039
Author(s):  
Robin Harrington ◽  
Biswajit Das ◽  
Tingting Jiang ◽  
Jennifer S. LoCoco ◽  
Rajesh Patidar ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Residual Disease ◽  
Digital Pcr ◽  
Coding Region ◽  
Single Nucleotide Variants ◽  
Liquid Biopsies ◽  
Driver Genes ◽  
Cell Free Dna ◽  
Initial Validation ◽  
Free Dna

3039 Background: Liquid biopsies are emerging as a powerful complement to tumor biopsies for the clinical management of cancer patients. A large gene panel with robust analytical performance that accurately assesses variants, tumor mutational burden (TMB), and microsatellite instability in plasma would be of high value for immunotherapy studies, monitoring minimal residual disease and early cancer detection. To this end, we have completed the initial validation for the cell-free DNA (cfDNA) assay, TruSight Oncology 500 (TSO500), which interrogates the full coding region of 523 genes plus selected intronic regions for fusion detection in 23 driver genes. Methods: Cell-free DNA was extracted from plasma collected from Streck or EDTA blood tubes and quantitated to achieve an assay input of ≥10 ng. Libraries were constructed using unique molecular identifiers (UMIs) and duplex barcodes for error correction, then enriched by target capture and sequenced on a NovaSeq 6000. Healthy donor (HD) specificity assessment used matched white blood cell results to filter germline and clonal hematopoiesis variants. Contrived specimens were used to evaluate sensitivity. Single nucleotide variants (SNVs) (n = 36), insertion/deletions (indels) (n = 19), copy number variants (CNVs) (n = 6), and fusions (n = 5) were tested in 2 multi-site replicates. Results: Sensitivity of detection at 0.5% variant allele fraction (VAF) was > 95% and > 97% for SNVs and indels, respectively. All expected CNVs were identified at the targeted threshold of ≥1.3X change and showed strong correlation with matched digital PCR results. All fusions were identified at ≥0.4% VAF. Specificity in HD was > 99.99%. In 22 temporally matched tumor and blood samples from late-stage patients, 58% of all reportable mutations in tumor were identified in cfDNA. Preliminary TMB analysis identified one TMB high case with POLE p.P286R observed in both tissue and cfDNA. Conclusions: In this initial validation study the TSO500 cfDNA assay exhibited high sensitivity and specificity consistent with requirements for clinical applications. Ongoing studies will further evaluate TSO500 as a complement or potential alternative to tissue biopsy for the genomic profiling of cancer patients.

scholarly journals DNA methylation fingerprint for the diagnosis and monitoring of hepatocellular carcinoma from tissue and liquid biopsies

2021 ◽  
Author(s):  
Emanuel Goncalves ◽  
Maria Reis ◽  
Jose B Pereira-Leal ◽  
Joana Cardoso
Keyword(s):  
Hepatocellular Carcinoma ◽  
Dna Methylation ◽  
Risk Score ◽  
False Negative ◽  
Great Promise ◽  
Liquid Biopsies ◽  
Cell Free Dna ◽  
Invasive Approach ◽  
Early Cancer Diagnosis ◽  
Free Dna

Hepatocellular carcinoma (HCC) is amongst the cancers with highest mortality rates and is the most common malignancy of the liver. Early detection is vital to provide the best treatment possible and liquid biopsies combined with analysis of circulating tumour DNA methylation show great promise as a non-invasive approach for early cancer diagnosis and monitoring with low false negative rates. To identify reliable diagnostic biomarkers of early HCC, we performed a systematic analysis of multiple hepatocellular studies and datasets comprising >1,500 genome-wide DNA methylation arrays, to define a methylation signature predictive of HCC in both tissue and cell-free DNA liquid biopsy samples. Our machine learning pipeline identified differentially methylated regions in HCC, some associated with transcriptional repression of genes related with cancer progression, that benchmarked positively against independent methylation signatures. Combining our signature of 38 DNA methylation regions, we derived a HCC detection score which confirmed the utility of our approach by identifying in an independent dataset 96% of HCC tissue samples with a precision of 98%, and most importantly successfully separated cfDNA of tumour samples from healthy controls. Notably, our risk score could identify cell-free DNA samples from patients with other tumours, including colorectal cancer. Taken together, we propose a comprehensive HCC DNA methylation fingerprint and an associated risk score for the early diagnosis and early relapse detection of HCC from liquid biopsies.

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