In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics

// Ida Grundberg 1,2 , Sara Kiflemariam 1 , Marco Mignardi 1,3 , Juliana Imgenberg-Kreuz 1,4 , Karolina Edlund 1,5 , Patrick Micke 1 , Magnus Sundström 1 , Tobias Sjöblom 1 , Johan Botling 1* and Mats Nilsson 1,3* 1 Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory. 2 Present address: Olink Bioscience, Uppsala Science Park, Uppsala, Sweden. 3 Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University. 4 Present address: Department of Medical Sciences, Uppsala University, Uppsala, Sweden. 5 Present address: Leibniz Research Centre for Working Environment and Human Factors (IfADo) at Dortmund TU, Dortmund, Germany. * Authors contributed equally to the work Correspondence: Mats Nilsson, email: // Keywords : Padlock probes, RCA, in situ, KRAS, cancer diagnostics Received : October 28, 2013 Accepted : November 19, 2013 Published : November 21, 2013 Abstract Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR , KRAS and TP53 . This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.