Flexible lab-tailored cut-offs for suitability of formalin-fixed tumor samples for diagnostic mutational analyses

Sara Mariani, Cristiana Di Bello, Lisa Bonello, Fabrizio Tondat, Donatella Pacchioni, Luca Molinaro, Antonella Barreca, Luigia Macrì, Luigi Chiusa, Paola Francia Di Celle, Paola Cassoni, Anna Sapino

Research output: Contribution to journalArticlepeer-review

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 cutoffs 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.

Original languageEnglish
Article numbere0121815
JournalPLoS One
Volume10
Issue number4
DOIs
Publication statusPublished - Apr 6 2015

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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