title: Applicability of a System for fully automated nucleic acid extraction from formalin-fixed paraffin-embedded sections for routine KRAS mutation testing.
abstract: Due to the approval of various new targeted therapies for the treatment of cancer, molecular pathology laboratories with a diagnostic focus have to meet new challenges: simultaneous handling of a large number of samples, small amounts of input material, and fragmentation of nucleic acids because of formalin fixation. As a consequence, fully automated systems for a fast and standardized extraction of high-quality DNA from formalin-fixed paraffin-embedded (FFPE) tissues are urgently needed. In this study, we tested the performance of a fully automated, high-throughput method for the extraction of nucleic acids from FFPE tissues. We investigated the extraction performance in sections of 5 different tissue types often analyzed in routine pathology laboratories (cervix, colon, liver, lymph node, and lung; n=340). Furthermore, we compared the quality, labor input, and applicability of the method for diagnostic purposes with those of a laboratory-validated manual method in a clinical setting by screening a set of 45 colorectal adenocarcinoma for the KRAS mutation. Automated extraction of both DNA and RNA was successful in 339 of 340 FFPE samples representing 5 different tissue types. In comparison with a conventional manual extraction protocol, the method showed an overall agreement of 97.7% (95% confidence interval, 88.2%-99.9%) for the subsequent mutational analysis of the KRAS gene in colorectal cancer samples. The fully automated system is a promising tool for a simple, robust, and rapid extraction of DNA and RNA from formalin-fixed tissue. It ensures a standardization of sample processing and can be applied to clinical FFPE samples in routine pathology.
authors.affiliation: Institute of Pathology, Charité University Hospital, Berlin, Germany. annika.lehmann@charite.de
authors.forename: Annika
authors.initials: A
authors.lastname: Lehmann
authors.forename: Christiane
authors.initials: C
authors.lastname: Schewe
authors.forename: Guido
authors.initials: G
authors.lastname: Hennig
authors.forename: Carsten
authors.initials: C
authors.lastname: Denkert
authors.forename: Wilko
authors.initials: W
authors.lastname: Weichert
authors.forename: Jan
authors.initials: J
authors.lastname: Budczies
authors.forename: Manfred
authors.initials: M
authors.lastname: Dietel
mesh_terms: D000230:Adenocarcinoma / Q000737:chemistry / Q000235:genetics; D057205:Automation, Laboratory; D002584:Cervix Uteri / Q000737:chemistry; D003106:Colon / Q000737:chemistry; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004252:DNA Mutational Analysis; D005260:Female; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D000071816:Glycodelin; D006023:Glycoproteins / Q000235:genetics; D006801:Humans; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008198:Lymph Nodes / Q000737:chemistry; D008297:Male; D025202:Molecular Diagnostic Techniques; D016612:Paraffin Embedding; D011257:Pregnancy Proteins / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000235:genetics; D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics*
chemical_list: D005404:Fixatives; D000071816:Glycodelin; D006023:Glycoproteins; C117307:KRAS protein, human; C045246:PAEP protein, human; D011257:Pregnancy Proteins; D011518:Proto-Oncogene Proteins; C484101:RPL37A protein, human; D012269:Ribosomal Proteins; D005557:Formaldehyde; D012313:RNA; D004247:DNA; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins