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Expression and Mutation Analyses of Candidate Cancer Genes In Situ
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cancers display heterogeneity in genetic profiles of the individual cancer cells and in the composition of different malignant and non-malignant cell populations. Such intra-tumor heterogeneity plays a role in treatment response and the emergence of resistance to cancer therapies. Approaches that address this complexity and improve stratification of patients for treatment are therefore highly warranted. Thus, the aims of this thesis were to further develop and apply in situ technologies for expression and mutation analyses of candidate cancer genes to gain a deeper understanding of cancer biology and to study intra-tumor heterogeneity.

In paper I, we established and validated a procedure for scalable in situ hybridization of large gene sets in human formalin-fixed paraffin-embedded tissues for analysis of gene expression. This method was used in paper II for large-scale expression analysis of the tyrosine kinome and phosphatome, two gene families whose members are frequently mutated in many forms of cancers. Systematic, compartment-specific expression mapping at cell type resolution enabled us to identify several novel vascular markers that have gone unnoticed in bulk transcriptomic analyses. In papers III and IV, we used padlock probes for in situ mutation detection in single cells for studies of genetic intra-tumor heterogeneity. In paper III, multiplex detection and genotyping of oncogenic point mutations was demonstrated in routinely processed tissue materials, whereas in paper IV we further the application by demonstrating multiplex detection of fusion gene transcripts.

Collectively, the work presented in this thesis employs in situ-based methods to obtain spatial resolution of gene expression and mutation patterns in normal and cancer tissues, thereby broadening our understanding of the cancer genome.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 50 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 842
Keyword [en]
cancer, in situ, in situ hybridization, padlock probes, mRNA, tyrosine kinases, tyrosine phosphatases, fusion transcripts
National Category
Genetics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-184510ISBN: 978-91-554-8546-7 (print)OAI: oai:DiVA.org:uu-184510DiVA: diva2:565577
Public defence
2012-12-21, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2012-11-30 Created: 2012-11-07 Last updated: 2013-02-11Bibliographically approved
List of papers
1. Scalable In Situ Hybridization on Tissue Arrays for Validation of Novel Cancer and Tissue-Specific Biomarkers
Open this publication in new window or tab >>Scalable In Situ Hybridization on Tissue Arrays for Validation of Novel Cancer and Tissue-Specific Biomarkers
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 3, e32927- p.Article in journal (Refereed) Published
Abstract [en]

Tissue localization of gene expression is increasingly important for accurate interpretation of large scale datasets from expression and mutational analyses. To this end, we have (1) developed a robust and scalable procedure for generation of mRNA hybridization probes, providing >95% first-pass success rate in probe generation to any human target gene and (2) adopted an automated staining procedure for analyses of formalin-fixed paraffin-embedded tissues and tissue microarrays. The in situ mRNA and protein expression patterns for genes with known as well as unknown tissue expression patterns were analyzed in normal and malignant tissues to assess procedure specificity and whether in situ hybridization can be used for validating novel antibodies. We demonstrate concordance between in situ transcript and protein expression patterns of the well-known pathology biomarkers KRT17, CHGA, MKI67, PECAM1 and VIL1, and provide independent validation for novel antibodies to the biomarkers BRD1, EZH2, JUP and SATB2. The present study provides a foundation for comprehensive in situ gene set or transcriptome analyses of human normal and tumor tissues.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-174583 (URN)10.1371/journal.pone.0032927 (DOI)000303062000022 ()
Available from: 2012-05-29 Created: 2012-05-22 Last updated: 2017-12-07Bibliographically approved
2. Expression analysis at cell type resolution of the tyrosine kinome and phosphatome in human normal and malignant tissues
Open this publication in new window or tab >>Expression analysis at cell type resolution of the tyrosine kinome and phosphatome in human normal and malignant tissues
(English)Manuscript (preprint) (Other academic)
National Category
Genetics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-184501 (URN)
Available from: 2012-11-07 Created: 2012-11-07 Last updated: 2013-02-11
3. In situ mutation detection in cancer tissue sections for research and diagnostics in clinical oncology
Open this publication in new window or tab >>In situ mutation detection in cancer tissue sections for research and diagnostics in clinical oncology
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(English)Article in journal (Other academic) Submitted
National Category
Genetics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-184506 (URN)
Available from: 2012-11-07 Created: 2012-11-07 Last updated: 2013-02-11
4. Direct detection of TMPRSS2-ERG fusion transcripts in prostate cancer tissues by padlock probes
Open this publication in new window or tab >>Direct detection of TMPRSS2-ERG fusion transcripts in prostate cancer tissues by padlock probes
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(English)Manuscript (preprint) (Other academic)
National Category
Genetics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-184507 (URN)
Available from: 2012-11-07 Created: 2012-11-07 Last updated: 2013-02-11

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