Ffpe tissue kit

Sample processing from tissue or buffy coat, library preparation, hybrid capture, and sequencing were performed at Personal Genome Diagnostics Inc. (Baltimore, MD)^{15 (link)}. Briefly, DNA was extracted from FFPE tissue and matched-normal buffy coat cells using the Qiagen FFPE Tissue Kit and DNA Blood Mini Kit, respectively (Qiagen, Hilden, Germany; catalog numbers 56404 and 51104, respectively). Genomic DNA was sheared using a Covaris sonicator (Woburn, MA, USA) to a size range of 150–450 bp, and subsequently used to generate a genomic library using the New England Biolabs (Ipswich, MA, USA) end-repair, A-tailing, and adapter ligation modules (catalog numbers E6050, E6053, and E6056, respectively). Finally, genomic libraries were amplified and captured using the Agilent SureSelect XT in-solution hybrid capture system with a 120 bp RNA panel targeting the pre-defined regions of interest across full exonic regions. Captured libraries were sequenced on the Illumina HiSeq 2000 or 2500 (Illumina, San Diego, CA, USA) with 100-bp paired-end reads.

DNA extraction and STR analyses were by the Denver Police Department Crime Laboratory DNA Unit using validated forensic standard operating procedures [16 (link),17 (link)]. Samples were collected into GeneAmp thin-walled reaction tubes (0.5 ml; Applied Biosystems, Carlsbad, CA, USA). Lymphocyte DNA was extracted manually using the FFPE Tissue kit (Qiagen in USA). Total human and male DNA were assessed with the Quantifiler Trio DNA Quantification Kit (Applied Biosystems, Carlsbad, CA, USA). DNA was extracted using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE Tissues (Applied Biosystems, Carlsbad, CA, USA). The number of tumor cells microdissected per sample was automatically recorded by the Arcturus XT system.

In the initial clinical trial protocol that was developed in the year 2009, MGMT testing was not established as standard procedure for patients included in the trial; it was added ex post. A retrospective collection of samples for MGMT analysis was thus only successful in a fraction of patients (see Section 2. Results). Depending on availability, DNA was extracted from either tumor tissue or formalin-fixed paraffin-embedded tissue (FFPE) sections using the QiAmp DNA Mini-Kit and FFPE tissue kit (both Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Subsequently, DNA modification was performed with the EpiTect Bisulfite Kit (Qiagen). Using the Therascreen MGMT Pyro Kit (Qiagen), bisulfite converted genomic DNA was amplified by PCR and sequenced on a Pyromark Q24 (Qiagen, Germany) system. Following previous research [58 (link)], a mean value of the methylation percentage of the four CpG sites investigated was calculated. Accordingly, a mean methylation >8% was considered as methylated.

Tumor DNA extracted from FFPE tumor tissue (FFPE Tissue Kit, Qiagen S.r.l., Milano, Italy) was amplified using specific primers for exon 4 of IDH1 and IDH2 genes. (IDH1: forward primer 5’-TGTAAAACGACGGCCAGTGGATGCTGCAGAAGCTATAA-3’; reverse primer 5’-CAGGAAACAGCTATGACCTTCATACCTTGCTTAATGGG-3’. IDH2: forward primer 5’-TGTAAAACGACGGCCAGTAATTTTAGGACCCCCGTCTG-3’; reverse primer 5’-CAGGAAACAGCTATGACCGGGGTGAAGACCATTTTGAA-3’). Codon 100 and 132 of IDH1 and codon 140 and 172 of IDH2 were then analyzed by Sanger sequencing method [14 (link)].

Sample processing from tissue, library preparation, hybrid capture, and sequencing were performed at Personal Genome Diagnostics Inc. (Baltimore, MD)^{15 (link)}. Briefly, DNA was extracted from FFPE tissue using the Qiagen FFPE Tissue Kit (Qiagen, Hilden, Germany; catalog number 56404). Genomic libraries were prepared, amplified, and captured using PGDx elio tissue complete sample preparation kits. Captured libraries were sequenced on the Illumina NextSeq 500 or 550 (Illumina, San Diego, CA, USA) with 150 bp paired-end reads.

H&E-stained slides were marked by an expert gynaecological pathologist (CSH) to identify tumour areas suitable for macrodissection in order to enrich for tumour cellularity. DNA extraction was performed using the QIAamp DNA formalin-fixed paraffin-embedded (FFPE) Tissue Kit (Qiagen, Venlo, Netherlands) and Deparaffinization Solution according to the manufacturer’s instructions.