Mariani S, Di Bello C, Bonello L, Tondat F, Pacchioni D, Molinaro L, et al. (2015) Flexible Lab-Tailored Cut-Offs for Suitability of Formalin-Fixed Tumor Samples for Diagnostic Mutational Analyses. PLoS ONE 10(4): e0121815.

Sara Mariani1, Cristiana Di Bello2,4, Lisa Bonello3,4, Fabrizio Tondat3, Donatella Pacchioni2, Luca Molinaro2, Antonella Barreca1, Luigia Macrì2, Luigi Chiusa2, Paola Francia di Celle2, Paola Cassoni1, Anna Sapino1

1Department of Medical Sciences; University of Torino, Torino, Italy
2Azienda Ospedaliera Universitaria Città della Salute e della Scienza; Presidio Ospedaliero Molinette of Torino, Torino, Italy
3Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy
4Contributed equally to this work


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.

Read the complete publication at this link