Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Genetic profiling of tumours using both circulating free DNA and circulating tumour cells isolated from the same preserved whole blood sample.

Author(s): Rothwell DG, Smith N, Morris D, Leong HS, Li Y, Hollebecque A, Ayub M, Carter L, Antonello J, Franklin L, Miller C, Blackhall F, Dive C, Brady G

Publication: Mol Oncol, 2016, Vol. 10, Page 566-74

PubMed ID: 26639657 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of processing delays and tube type on the level of cell-free DNA (cfDNA) and on next-generation sequencing (NGS). The copy number profile of DNA from isolated CTCs, EDTA cfDNA, CellSave cfDNA, and WBCs were also compared. Blood specimens were collected from 20 healthy donors, 11 small cell lung cancer patients, and 34 melanoma patients into CellSave and EDTA Vacutainer tubes and transferred to the laboratory for processing. To test the effects of delayed processing, blood was stored in EDTA tubes (unspecified number of specimens) for 1, 24, 48, 72, or 96 h before processing. To test the effects of CellSave tubes, blood from the 20 healthy volunteers was collected in EDTA and CellSave tubes and centrifuged within 4 h (2-3.3 h) or after being sent through the postal system at ambient temperatures (93.3-95.3 h). To examine the effects of delayed analysis further, patient blood was stored at room temperature for 96 h in CellSave tubes and in EDTA tubes for less than 4 h. Plasma was obtained by two centrifugations at 2000 x g for 10 min and frozen at -80°C. DNA was isolated using the QIAamp Circulating Nucleic Acid Kit and quantified using the TaqMan RNAse P Detection Kit. cfDNA was sequenced using NEBNext Ultra DNA Library Prep Kit for Illumina and an Illumina miSeq with three technical replicates per specimen. CTCs and WBCs were obtained from the CellSearch cartridges in the CellSave tubes and were isolated using the DEParray system. Single cells were subjected to whole genome amplification using the Ampli1 WGA Kit and sequencing using the Qiagen GeneRead Lung Cancer v1 Panel.

   Summary of Findings:

   The level of RNAse P detected in plasma increased 3-fold and 60-fold when centrifugation of blood in EDTA tubes was delayed by 24 h and 96 h, respectively; however, the amount of RNAse P detected in plasma was the same in EDTA and CellSave tubes processed within 4 h and CellSave tubes processed after 4 days in the mail at ambient temperatures (healthy controls) or 4 days at room temperature (cancer patients). In contrast, the amount of RNAse P DNA detected in plasma was significantly higher than in CellSave tubes when the specimens were mailed in EDTA tubes (healthy controls), regardless of processing delay, and in EDTA tubes processed within 4 h (P<0.001). Mapability, overall coverage, percentage of duplicate reads, mutation rates, and frequency of transition and transversion mutations were comparable for cfDNA from CellSave tubes processed after 96 h and EDTA tubes processed within 4 h. For cancer patients, identical mutations were identified in specimens from the two tube types. Copy number alteration profiles of DNA from isolated CTCs, EDTA cfDNA, CellSave cfDNA, and WBC DNA (germline control) showed clear and consistent changes in cfDNA and CTC specimens compared to WBC DNA.

   Biospecimens

   • Bodily Fluid - Plasma
   • Cell - White Cells
   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • None (Fresh)

   Diagnoses:

   • Normal
   • Neoplastic - Melanoma
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	EDTA Tube CellSave Tube
   Storage	Storage duration	24 h 48 h 72 h 96 h <4 h 4 days 1 h
   Storage	Between site transportation method	Mailed Not transported

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