DNA and RNA extraction and quantification
Germline DNA and RNA were extracted from peripheral blood using the
MagNA Pure Compact extractor system (Roche Diagnostics), according to
the manufacturer’s recommendations. The PAXgene Blood RNA Kit
(PreAnalytiX) was used to extract germline RNA when the patient could
not come to our hospital. Tumor DNA and RNA were extracted from
7µm-thick FFPE tissue sections, and a hematoxylin and eosin-stained
section of each block allowed the assessment of tumor cell area and
content by two experienced pathologists. Tumor DNA was purified using
the QIAamp DNA FFPE Tissue Kit from Qiagen, while tumor RNA was isolated
employing the RNeasy FFPE Kit (Qiagen), according to their corresponding
protocols. The NanoDrop ND-1000 Spectrophotometer was used to assess the
DNA and RNA quantity and quality. However, for NGS purposes, the quality
of DNA samples was also tested by agarose gel electrophoresis and the
concentration of the samples was measured in a Qubit 3.0 Fluorometer
(Life Technologies) and a 2100 Bioanalyzer (Agilent).