Biospectrometer plus

Genomic DNA was extracted from tumor sections using the QIAamp Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions, and quantified on a BioSpectrometer Plus (Eppendorf, Hamburg, Germany). The entire coding (exons 2-11) and promoter regions of P53, were amplified by standard PCR protocols using Taq DNA Polymerase (Thermo Scientific, USA) and the primer pairs with sequences available on request. Direct sequencing was performed using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's instructions on an ABI 310 Genetic Analyzer (Applied Biosystems). The sequence analysis software Alamut® (Interactive Biosoftware) was used to interpret variants.

Genomic DNA was extracted from peripheral blood cells (PBCs) and tissue samples using the QIAamp Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, and quantified on a BioSpectrometer Plus (Eppendorf, Hamburg, Germany). The entire coding regions of PIK3CA (RefSeq NM_006218.2), including all splice junctions and adjacent intronic sequences were amplified by standard PCR protocols using the AmpliTaq Gold DNA Polymerase (Applied Biosystems, London, UK) and the primer pairs listed in Table A in S1 File. Direct sequencing was performed using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s instructions on an ABI 310 Genetic Analyzer (Applied Biosystems).

Genomic DNA was extracted from biopsy sections derived from PCNSL specimens using the QIAamp Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions, and quantified on a BioSpectrometer Plus (Eppendorf, Hamburg, Germany). The entire coding regions of Stat3 (RefSeq NM_139276.2), including all splice junctions and adjacent intronic sequences, were amplified by standard PCR protocols using Taq DNA Polymerase (Thermo Scientific, USA) and the primer pairs listed in Table 3. Direct sequencing was performed using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's instructions on an ABI 310 Genetic Analyzer (Applied Biosystems). The sequence analysis software Alamut® (Interactive Biosoftware) was used to interpret variants. Online databases including dbSNP (Database the Single Nucleotide Polymorphism Database), 1000 Genomes, ClinVar, EXAC (Exome Aggregation Consortium), COSMIC (Catalogue of Somatic Mutations in Cancer), ESP (Exome Sequencing Project), as well as on line search engines (e.g. PubMed, LOVD), were used to search for previously described variants.