The results of our study may be summarized as follows: TMNGS can be applied on DNA from routinely prepared paraffin tissues; the data 6-Acetamidohexanoic acid produced are quantitative and thus permit the description of the molecular subclonal composition of a tumor. The 2-Aminoheptane introduction of targeted drugs is changing the profile of information needed to plan a therapeutical approach that entails multiple lines of intervention. In this scenario, the histopathological diagnosis based on morphological classifications is no longer sufficient, and will need to be complemented by a comprehensive description of the specific molecular alterations and clonal heterogeneity of the tumor. Proof of concept reports have already shown the potential application of NGS techniques using DNA from FFPE tissues. However, its introduction in the clinical routine still needs validation of each step leading from the sample to results as well as the design of appropriate panels to specifically interrogate multiple tumor categories. All the 35 tumor samples in our representative series of upper gastrointestinal system cancers were characterized by at least one single specific molecular alteration among the 46 genes analyzed, some of which also represent a potential therapeutic target. Two or more mutations were found in 20/35 cases. Moreover, several genes were altered in more than one tumor type, suggesting the possibility of a molecular subclassification of tumors that crosses the borders of histology and puts the focus on molecular and potentially actionable alterations. While these commonly altered genes could be detected by the commercial assay used in the present work, additional crossborder molecular alterations or mutations that remain confined to a specific tumor class are being reported. For this reason, the design of specialized and optimized multigene panels will be the next mandatory step. Indeed, a European consortium of research centers has already developed a TMNGS panel specifically tailored to target colon and lung cancer.In conclusion, our study demonstrates the ability of TM-NGS to detect and quantitate multiple gene alterations, thus moving a further step towards a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and mutational analysis of multiple genes using routinely processed tissues.