Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Maass KK

PROVIDER: S-EPMC8232701 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Maass Kendra K KK Schad Paulina S PS Finster Agnes M E AME Puranachot Pitithat P Rosing Fabian F Wedig Tatjana T Schwarz Nathalie N Stumpf Natalie N Pfister Stefan M SM Pajtler Kristian W KW

Cancers 20210615 12

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 ...[more]

PMID: 34203921

Similar Datasets

Project description:Volumetric absorptive microsampling (VAMS) is a novel approach that allows single-drop (10 μL) blood collection. Integration of VAMS with mass spectrometry (MS)-based untargeted metabolomics is an attractive solution for both human and animal studies. However, to boost the use of VAMS in metabolomics, key pre-analytical questions need to be addressed. Therefore, in this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. We first evaluated the best extraction procedure for the polar metabolome and found that the highest number and amount of metabolites were recovered upon extraction with acetonitrile/water (70:30). In contrast, basic conditions (pH 9) resulted in divergent metabolite profiles mainly resulting from the extraction of intracellular metabolites originating from red blood cells. In addition, the prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but once the VAMS devices were stored at - 80 °C, the metabolome remained stable for up to 6 months. The time used for drying the sample did also affect the metabolome. In fact, some metabolites were rapidly degraded or accumulated in the sample during the first 48 h at room temperature, indicating that a longer drying step will significantly change the concentration in the sample. Graphical abstract Volumetric absorptive microsampling (VAMS) is a novel technology that allows single-drop blood collection and, in combination with mass spectrometry (MS)-based untargeted metabolomics, represents an attractive solution for both human and animal studies. In this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. The latter revealed that prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but if VAMS devices were stored at - 80 °C, the metabolome remained stable for up to 6 months.

Project description:The optimal treatment for patients with oligometastatic non-small cell lung cancer (NSCLC) remains unclear. Some patients with oligometastatic disease can experience prolonged remission after locally consolidative radiation therapy (RT), while others harbor micrometastatic disease (below current limits of detection by imaging) that may benefit from further prioritization of systemic therapy. To better risk-stratify this population and identify the patients most likely to benefit from locally consolidative radiation therapy, we performed a multi-institutional cohort study of patients with oligometastatic NSCLC undergoing liquid biopsy analysis of circulating tumor DNA (ctDNA). Among this real-world cohort of 1,487 patients undergoing analysis (using the Tempus xF assay), a total of 1,880 ctDNA liquid biopsies along with paired clinical data were obtained across various timepoints. Approximately 20% (n=309) of patients had ctDNA obtained prior to RT and after their diagnosis of oligometastatic disease. Samples were de-identified and analyzed for mutational burden and variant frequencies of detectable deleterious (or likely deleterious) mutations in plasma. Patients with undetectable ctDNA before RT had significantly improved progression-free survival and overall survival compared to patients with detectable ctDNA prior to RT. In patients that received RT, 598 pathogenic (or likely deleterious) variants were identified. ctDNA mutational burden pre-RT and ctDNA maximum variant allele frequency (VAF) pre-RT were both significantly inversely correlated with both progression-free (P = 0.0031 for mutational burden, P = 0.0084 for maximum VAF) and overall survival (P = 0.045 for mutational burden, P = 0.0073 for maximum VAF). Patients without detectable ctDNA prior to RT had significantly improved progression-free survival (P = 0.004) and overall survival (P = 0.03) compared to patients with detectable ctDNA prior to RT. These data suggest that in patients with oligometastatic NSCLC, pre-radiotherapy ctDNA analysis can potentially identify the patients most likely to benefit from locally consolidative RT and experience prolonged progression-free and overall survival. Similarly, ctDNA may be useful to identify those patients with undiagnosed micrometastatic disease, in whom it may be appropriate to prioritize systemic therapy.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets