title: A multiplex MALDI-TOF MS approach facilitates genotyping of DNA from formalin-fixed paraffin-embedded tumour specimens.
abstract: OBJECTIVE
  The impact of single-nucleotide polymorphisms (SNPs) on tumour susceptibility and pathogenesis has gained enormous attention. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based genotyping facilitates the analysis of short DNA amplicons and is, therefore, a promising tool for the investigation of formalin-fixed paraffin-embedded (FFPE) tissue samples, particularly in targeted genotyping analysis.
  
  
  METHODS
  To examine the applicability of genotyping FFPE DNA with MALDI-TOF MS in multiplex reactions, we investigated five DNA samples extracted from FFPE tumour specimens from follicular lymphoma patients using different extraction methods (phenol-chloroform, commercial kit). Thirty-one SNPs from 25 genes, integrated in different-sized multiplex assays (7-plex, 10-plex, 14-plex, 24-plex), were analyzed. To investigate the reliability of genotyping tumour-derived DNA extracted from FFPE tissue, we examined 64 FFPE tumour specimens in comparison with matched germline DNA samples.
  
  
  RESULTS
  Call rates of 99.6 (274/275) and 93.5% (257/275) were observed for the DNA extracted with the phenol-chloroform approach or the commercial extraction kit, respectively. Increasing the number of SNPs per assay resulted in reduced genotyping call rates and genotyping quality, especially in the DNA samples isolated with the commercial extraction kit. When comparing the genotypes of DNA derived from germline and tumour (FFPE) specimens, a perfect concordance rate of 100% was detected.
  
  
  CONCLUSIONS
  Our data delineate that MALDI-TOF-based genotyping of FFPE DNA is reliable and reproducible even in multiplex reactions, enabling the retrospective investigation of FFPE study cohorts in future experiments.
authors.affiliation: Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany. heike.horn@ikp-stuttgart.de
authors.forename: Heike
authors.initials: H
authors.lastname: Horn
authors.forename: Christiane
authors.initials: C
authors.lastname: Pott
authors.forename: Jörg
authors.initials: J
authors.lastname: Kalla
authors.forename: Martin
authors.initials: M
authors.lastname: Dreyling
authors.forename: Andreas
authors.initials: A
authors.lastname: Rosenwald
authors.forename: German
authors.initials: G
authors.lastname: Ott
authors.forename: Matthias
authors.initials: M
authors.lastname: Schwab
authors.forename: Elke
authors.initials: E
authors.lastname: Schaeffeler
mesh_terms: D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D005557:Formaldehyde / Q000737:chemistry*; D005838:Genotype; D006801:Humans; D009369:Neoplasms / Q000097:blood / Q000235:genetics*; D016612:Paraffin Embedding / Q000379:methods*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D004273:DNA, Neoplasm; D005557:Formaldehyde