title: Rapid targeted somatic mutation analysis of solid tumors in routine clinical diagnostics.
abstract: Tumor genotyping is an essential step in routine clinical practice and pathology laboratories face a major challenge in being able to provide rapid, sensitive and updated molecular tests. We developed a novel mass spectrometry multiplexed genotyping platform named PentaPanel to concurrently assess single nucleotide polymorphisms in 56 hotspots of the 5 most clinically relevant cancer genes, KRAS, NRAS, BRAF, EGFR and PIK3CA for a total of 221 detectable mutations. To both evaluate and validate the PentaPanel performance, we investigated 1025 tumor specimens of 6 different cancer types (carcinomas of colon, lung, breast, pancreas, and biliary tract, and melanomas), systematically addressing sensitivity, specificity, and reproducibility of our platform. Sanger sequencing was also performed for all the study samples. Our data showed that PentaPanel is a high throughput and robust tool, allowing genotyping for targeted therapy selection of 10 patients in the same run, with a practical turnaround time of 2 working days. Importantly, it was successfully used to interrogate different DNAs isolated from routinely processed specimens (formalin-fixed paraffin embedded, frozen, and cytological samples), covering all the requirements of clinical tests. In conclusion, the PentaPanel platform can provide an immediate, accurate and cost effective multiplex approach for clinically relevant gene mutation analysis in many solid tumors and its utility across many diseases can be particularly relevant in multiple clinical trials, including the new basket trial approach, aiming to identify appropriate targeted drug combination strategies.
authors.affiliation: Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Gilda
authors.initials: G
authors.lastname: Magliacane
authors.affiliation: Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Greta
authors.initials: G
authors.lastname: Grassini
authors.affiliation: Diatech Pharmacogenetics Company, Jesi, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Bartocci
authors.affiliation: Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Francaviglia
authors.affiliation: Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Dal Cin
authors.affiliation: Diatech Pharmacogenetics Company, Jesi, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Barbieri
authors.affiliation: Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Arrigoni
authors.affiliation: Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Lorenza
authors.initials: L
authors.lastname: Pecciarini
authors.affiliation: Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Doglioni
authors.affiliation: Unit of Pathology, IRCCS San Raffaele Scientific Institute, Milano, Italy.
authors.forename: Maria Giulia
authors.initials: MG
authors.lastname: Cangi
mesh_terms: D001483:Base Sequence; D058534:Class I Phosphatidylinositol 3-Kinases; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics; D060005:Genotyping Techniques / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D015203:Reproducibility of Results; D012189:Retrospective Studies; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: C117307:KRAS protein, human; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C512478:EGFR protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords: Pathology Section; high throughput mass-spectrometry; molecular diagnostics; mutations; solid tumors; target therapy