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In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
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2013 (English)In: OncoTarget, ISSN 1949-2553, Vol. 4, no 12, 2407-2418 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2013. Vol. 4, no 12, 2407-2418 p.
Keyword [en]
Padlock probes, RCA, in situ, KRAS, cancer diagnostics
National Category
Cell Biology
URN: urn:nbn:se:su:diva-100118ISI: 000328936800022OAI: diva2:691354


Available from: 2014-01-27 Created: 2014-01-27 Last updated: 2014-12-05Bibliographically approved
In thesis
1. In situ Sequencing: Methods for spatially-resolved transcriptome analysis
Open this publication in new window or tab >>In situ Sequencing: Methods for spatially-resolved transcriptome analysis
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is well known that cells in tissues display a large heterogeneity in gene expression due to differences in cell lineage origin and variation in the local environment at different sites in the tissue, a heterogeneity that is difficult to study by analyzing bulk RNA extracts from tissue. Recently, genome-wide transcriptome analysis technologies have enabled the analysis of this variation with single-cell resolution. In order to link the heterogeneity observed at molecular level with the morphological context of tissues, new methods are needed which achieve an additional level of information, such as spatial resolution.

In this thesis I describe the development and application of padlock probes and rolling circle amplification (RCA) as molecular tools for spatially-resolved transcriptome analysis. Padlock probes allow in situ detection of individual mRNA molecules with single nucleotide resolution, visualizing the molecular information directly in the cell and tissue context. Detection of clinically relevant point mutations in tumor samples is achieved by using padlock probes in situ, allowing visualization of intra-tumor heterogeneity. To resolve more complex gene expression patterns, we developed in situ sequencing of RCA products combining padlock probes and next-generation sequencing methods. We demonstrated the use of this new method by, for the first time, sequencing short stretches of transcript molecules directly in cells and tissue. By using in situ sequencing as read-out for multiplexed padlock probe assays, we measured the expression of tens of genes in hundreds of thousands of cells, including point mutations, fusions transcripts and gene expression level.

These molecular tools can complement genome-wide transcriptome analyses adding spatial resolution to the molecular information. This level of resolution is important for the understanding of many biological processes and potentially relevant for the clinical management of cancer patients.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 49 p.
Padlock probes, rolling circle amplification, in situ sequencing, spatially-resolved transcriptomics, molecular diagnostics
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
urn:nbn:se:su:diva-110057 (URN)978-91-7649-066-2 (ISBN)
Public defence
2015-01-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2014-12-29 Created: 2014-12-05 Last updated: 2014-12-15Bibliographically approved

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