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Finzel et al. J Cancer Metastasis Treat 2018;4:21 I http://dx.doi.org/10.20517/2394-4722.2018.10 Page 3 of 10

prostate (6.3%), hepatobiliary (5.7%), brain (5.1%), pancreatic (5.1%), gastric (4.6%), carcinoma of unknown
primary (3.4%), head and neck cancer (1.7%) and other cancers (11.1%). Solid biopsies were obtained as
a formalin-fixed paraffin-embedded (FFPE) block from either the primary or the metastatic tumor and
underwent review by a pathologist to determine if the criteria for sample acceptance were met: tumor tissue
2
> 10% of the whole sample and tumor size > 5 mm in order to have enough tumor material, and lymphocyte
invasion < 20% in the region where the tumor cells were located to avoid lymphocyte DNA contamination. In
addition, tissue samples were macro-dissected to remove contaminating normal tissue. Blood was collected
in two Cell-Free DNA BCT® CE tubes (Streck, La Vista, USA) and underwent visual inspection to determine
that no hemolysis had occurred.

Sample preparation and next-generation sequencing
DNA was extracted from FFPE tissue and cfDNA was extracted from blood using the Qiagen DNA
FFPE Tissue Kit or Qiagen DNA Blood Mini Kit (Qiagen, Valencia, USA) respectively. DNA quantity was
measured using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, USA). To identify somatic
alterations in tumor samples, we used our proprietary solution OncoSTRAT&GO™ (OncoDNA, Gosselies,
Belgium). This solution is built based on the Oncomine Comprehensive Panel v2 designed by ThermoFisher,
which we updated in order to analyse genome regions or genes not included in that version but shown
to be important in cancer. OncoSTRAT&GO™ is based on AmpliSeq technology, amplifying for next-
generation sequencing (NGS) whole exons and hotspot mutations of 192 genes (gene panel for the solid
biopsy part of OncoSTRAT&GO™) or hotspot mutations of 27 genes (gene panel for the liquid biopsy part
of OncoSTRAT&GO™). Briefly, the targeted sequencing libraries were generated using the Ion AmpliSeq
Library kit 2.0 according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, USA).
The starting material consisted of 10 ng DNA from FFPE or blood samples per pool of amplification (4 for
the solid part and 2 for the liquid part, respectively). The primers used for amplification were partially
digested by Pfu enzyme. The product of digestion was then ligated with corresponding barcoded adapters
and purified using Ampure Beads (Beckman Coulter Inc., Indianapolis, USA). The product of purification
was amplified for 5 more cycles and subsequently re-purified using Ampure Beads to generate the library
sample. The quality of the libraries was assessed using the Qubit dsDNA HS Assay kit (Thermo Fisher
Scientific, Waltham, USA). Ten pmol/L of each library was loaded into the IonChef system (Thermo Fisher
Scientific, Waltham, USA) for the emulsion polymerase chain reaction. Libraries were then loaded into the
sequencing chip that was placed in either the Personal Genome Machine, the Proton or the 5XL device
(Thermo Fisher Scientific, Waltham, USA) depending on the required throughput. An average coverage of
1000× was generated in order to detect single-base substitutions down to 5% for the FFPE, and of 10000× to
detect base substitutions down to 0.1% for blood samples.

Primary processing of next-generation sequencing data and identification of putative somatic
mutations
The data generated from the FFPE and blood samples were first aligned to the human reference sequence and
annotated using the Consensus Coding DNA Sequences, RefSeq, and Ensembl databases. NGS data were
then analysed using the Torrent Suite Software (Thermo Fisher Scientific, Waltham, USA). Next, somatic
mutations were identified with the Variant Caller 4.0 software (Thermo Fisher Scientific, Waltham, USA)
using the somatic high stringency parameters to ensure sufficient coverage of the analysed bases and to
exclude mapping and sequencing errors [Supplementary Tables 1 and 2]. Genetic aberration analysis was
focussed on single-base substitutions, small insertions and deletions. Candidate somatic alterations were
further filtered based on: coverage of > 100 in solid biopsy analysis; a forward-reverse ratio of 10%, 90%; the
exclusion of intronic and silent changes; and the retention of mutations resulting in missense mutations,
nonsense mutations, frameshifts, or splice site alterations in the protein coding region. A manual visual
inspection step was used to further remove artefactual changes.