Slides stained for an individual chromogen only were used to make a spectral library. The spectral library was used for computational segregation of the individual image components using the NuanceTM 3.0.2 software as described. After spectral unmixing, pseudo-colors were assigned to unmixed images, and composite images showing co-localization were generated with the Nuance 3.0.2 software.AnimalsAll animal work was approved by the neighborhood regulatory authority of Maastricht University and in accordance with EU and Dutch government laws and guidelines. Mouse experiments performed in Cincinnati complied with approved Institutional Animal Care and Use Committee guidelines and the rules of the Association for Assessment and Accreditation of Laboratory Animal Care International.Evaluation for rearrangements had not been performed. Laboratory Studies Expression plasmids containing mTOR complementary DNA were generated, and site-directed mutagenesis, stable transfections, analysis of cell-development inhibition, and immunoblot research were performed. For information, see the Methods section in Supplementary Appendix 1. Outcomes Mutations in MTOR and TSC2 The pretreatment tumor contained a somatic non-sense mutation in the tumor-suppressor gene TSC2 that inactivates the gene, enabling activation of the mTOR pathway7,8 and leading to sensitivity to mTOR inhibition in a few cancers1,2,4,9 .8,10 Somatic mutations in TSC2 have been identified in many tumor types, including cancers of the kidney and bladder as well as hamartomas and malignant perivascular epithelioid-cell tumors in individuals with tuberous sclerosis complex.