title: Flexible lab-tailored cut-offs for suitability of formalin-fixed tumor samples for diagnostic mutational analyses.
abstract: The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity.
authors.affiliation: Department of Medical Sciences; University of Torino, Torino, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Mariani
authors.affiliation: Azienda Ospedaliera Universitaria Città della Salute e della Scienza; Presidio Ospedaliero Molinette of Torino, Torino, Italy.
authors.forename: Cristiana
authors.initials: C
authors.lastname: Di Bello
authors.affiliation: Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.
authors.forename: Lisa
authors.initials: L
authors.lastname: Bonello
authors.affiliation: Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Tondat
authors.affiliation: Azienda Ospedaliera Universitaria Città della Salute e della Scienza; Presidio Ospedaliero Molinette of Torino, Torino, Italy.
authors.forename: Donatella
authors.initials: D
authors.lastname: Pacchioni
authors.affiliation: Azienda Ospedaliera Universitaria Città della Salute e della Scienza; Presidio Ospedaliero Molinette of Torino, Torino, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Molinaro
authors.affiliation: Department of Medical Sciences; University of Torino, Torino, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Barreca
authors.affiliation: Azienda Ospedaliera Universitaria Città della Salute e della Scienza; Presidio Ospedaliero Molinette of Torino, Torino, Italy.
authors.forename: Luigia
authors.initials: L
authors.lastname: Macrì
authors.affiliation: Azienda Ospedaliera Universitaria Città della Salute e della Scienza; Presidio Ospedaliero Molinette of Torino, Torino, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Chiusa
authors.affiliation: Azienda Ospedaliera Universitaria Città della Salute e della Scienza; Presidio Ospedaliero Molinette of Torino, Torino, Italy.
authors.forename: Paola Francia
authors.initials: PF
authors.lastname: di Celle
authors.affiliation: Department of Medical Sciences; University of Torino, Torino, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Cassoni
authors.affiliation: Department of Medical Sciences; University of Torino, Torino, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Sapino
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology*; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000473:pathology*; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D016612:Paraffin Embedding / Q000379:methods / Q000592:standards*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics
chemical_list: D005404:Fixatives; C117307:KRAS protein, human; D005557:Formaldehyde; C512478:EGFR protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras)
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