Extracellular Vesicle DNA Extraction and Sequencing in Ancient Serum Samples From Patients With Breast Cancer.
Author(s): Heidinger M, Egle D, Piscuoglio S, Navarro-Aguadero MÁ, Sánchez S, Hergueta-Redondo M, Gallardo M, Barrio S, García-Peláez B, Molina-Vila MA, Maggi N, Eller RS, Loesch JM, Alborelli I, Peinado H, Weber W, Weber WP
Publication: Anticancer Res, 2024, Vol. 44, Page 2981-2988
PubMed ID: 38925824 PubMed Review Paper? No
Purpose of Paper
This paper investigated the feasibility of extraction and mutation detection by next- generation sequencing (NGS) of cell-free DNA (cfDNA) and extracellular vesicle DNA (EV-DNA) from serum specimens stored for 30 to 38 years. Serum was collected from breast cancer patients at different stages of diagnosis and treatment.
Conclusion of Paper
The median yield and concentration of cfDNA and EV-DNA were comparable to one another, and sequencing was successful for all specimens. While identical mutations were detected in cfDNA and EV-DNA from serum collected from two patients at or after confirmed distant metastasis, both serum specimens collected during or after adjuvant treatment had PIK3CA mutations detected in the cfDNA but no mutation detected in the EV-DNA; the serum specimen collected at primary diagnosis had a TP53 mutation detected in the EV-DNA but not the cfDNA. Despite these discrepancies, the authors conclude that, like cfDNA, EV-DNA from archival serum specimens can be sequenced and used to address clinically relevant questions.
Studies
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Study Purpose
This study investigated the feasibility of extraction and mutation detection by next-generation sequencing (NGS) of cell-free DNA (cfDNA) and extracellular vesicle DNA (EV-DNA) from serum specimens stored for 30 to 38 years. Blood was collected from twenty-seven patients with breast cancer and serum (28 specimens) was separated by centrifugation at 3075 x g for 20 min, aliquoted into cryogenic tubes, and stored at -70 to -80°C for 30-38 years, during which time they were transferred twice in “transportable refrigerating boxes” to avoid thaw. cfDNA was extracted from serum using the QIAsymphony DSP Virus/Pathogen Midi Kit and a QIAsymphony robot. EVs were isolated from serum using the Exo-Gag Kit and DNA was extracted using the QIAamp DNA Kit. DNA was quantified using the Qubit dsDNA HS Assay Kit. Mutations were detected in matched specimens of five patients using the Oncomine Breast cfDNA Research Assay v2 and an Ion S5 Sequencer. A mutation was called if the variant read fraction (VRF) was greater than the limit of detection and the experimental sensitivity (2([ng DNA]/0.00649)-1.
Summary of Findings:
The median yields and concentrations were similar for cfDNA and EV-DNA (26.1 ng versus 27.6 ng and 0.87 versus 0.69 ng/μl, respectively). Sequencing was successful in all specimens. cfDNA and EV-DNA from serum collected at or after confirmed distant metastasis showed no mutations in both DNA types from one patient and PIK3CA mutations in both DNA types from the other patient. Both patients with serum collected during or after adjuvant treatment had a PIK3CA mutation detected in the cfDNA but no mutation detected in the EV-DNA. In the patient from whom serum was collected at primary diagnosis, a TP53 mutation was detected in the EV-DNA but not the cfDNA. The authors conclude that, like cfDNA, EV-DNA from archival specimens can be sequenced and used to address clinically relevant questions
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Fluorometry DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method QIAsymphony DSP Virus/Pathogen Midi kit with a QIAsymphony robot
Exo-Gag kit for EV isolation and QIAamp DNA kit for DNA extraction
Biospecimen Aliquots and Components Blood and blood products Serum
Serum vesicles