Somatic Tumor Mutations Detected by Targeted Next Generation Sequencing in Minute Amounts of Serum-Derived Cell-Free DNA

Marjolein J A Weerts,Winand N M Dinjens,Ronald Van Marion,Jean C A Helmijr,Anita M A C Trapman-Jansen,Maurice P H M Jansen,Niels M G Krol,John W M Martens,Stefan Sleijfer,Corine M Beaufort

doi:10.1038/s41598-017-02388-7

Marjolein J A Weerts, Winand N M Dinjens + Show 8 more

Open Access

https://doi.org/10.1038/s41598-017-02388-7

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Journal: Scientific Reports	Publication Date: May 18, 2017
Citations: 7	License type: open-access

Affiliation: Erasmus MC Cancer Institute

Abstract

The use of blood-circulating cell-free DNA (cfDNA) as ‘liquid-biopsy’ is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment. In exploring cfDNA, valuable repositories are biobanks containing material collected over time, however these retrospective cohorts have restrictive resources. In this study, we aimed to detect tumor-specific mutations in only minute amounts of serum-derived cfDNA by using a targeted next generation sequencing (NGS) approach. In a retrospective cohort of ten metastatic breast cancer patients, we profiled DNA from primary tumor tissue (frozen), tumor-adjacent normal tissue (formalin-fixed paraffin embedded), and three consecutive serum samples (frozen). Our presented workflow includes comparisons with matched normal DNA or in silico reference DNA to discriminate germline from somatic variants, validation of variants through the detection in at least two DNA samples of an individual, and the use of public databases on variants. By our workflow, we were able to detect a total of four variants traceable as circulating tumor DNA (ctDNA) in the sera of three of the ten patients.

Highlights

The use of blood-circulating cell-free DNA as ‘liquid-biopsy’ is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment
We present a next generation sequencing (NGS) workflow using a custom 45-gene sequencing panel on the Ion Personal Genome Machine (PGM) system applied to a retrospective cohort of ten metastatic breast cancer patients
Using our custom panel we sequenced a total of forty-six DNA samples, derived from fresh frozen (FF) primary breast carcinoma specimens, FF serum and formalin-fixed and paraffin embedded (FFPE) tumor-adjacent normal mammary epithelial specimens

Summary

Introduction

The use of blood-circulating cell-free DNA (cfDNA) as ‘liquid-biopsy’ is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment. We aimed to detect tumor-specific mutations in only minute amounts of serum-derived cfDNA by using a targeted generation sequencing (NGS) approach. In exploring the potential of cfDNA in oncology, valuable repositories are biobanks containing material collected over time These retrospective cohorts have restrictive resources, including suboptimal specimen preservations such as formalin-fixed and paraffin embedded (FFPE) specimens or limiting amounts of DNA available for profiling, challenging the analysis of these cohorts. We present a NGS workflow using a custom 45-gene sequencing panel on the Ion PGM system applied to a retrospective cohort of ten metastatic breast cancer patients This workflow includes comparisons with FFPE matched normal DNA from tumor-adjacent histologically normal mammary epithelium or in silico reference DNA to discriminate germline from somatic variants. For all ten patients, we identified variants in their primary tumor DNA (standard DNA quantities) and three consecutive serum samples (minute DNA quantities)

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