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Diagnostic mutation testing in situ in routine FFPE tissue sections for treatment prediction in clinical oncology
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Molekylär Diagnostik)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. (Translational Tumor Pathology)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. (Translational Tumor Pathology)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. (Translational Tumor Pathology)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Activating mutations in the KRAS gene are present in different cancer types and are strongly associated with resistance to epidermal growth factor receptor (EGFR) inhibitor therapy. Hence there is a requirement for sensitive KRAS mutation analysis to determine the most suitable treatment for the patients. Also, little is known about the impact of tumor heterogeneity with regard to KRAS mutation status in different sub-clones during tumorigenesis, and if this is important for treatment response and prognosis. To improve the diagnostic accuracy, we developed an RNA-based genotyping assay that targets KRAS-mutations in codon 12 and 13 in situ on tissue samples by the use of multiple mutation specific padlock probes and rolling-circle amplification. Thus, the distribution of wild-type (green rolling-circle products) and mutated (red rolling-circle products) KRAS alleles can be determined for single cancer cells in different parts of a heterogeneous tumor without the use of microdissection. We demonstrate reliable detection of KRAS point mutations on cytologic tumor imprints as well as on fresh frozen and formalin-fixed paraffin-embedded tissue sections from colorectal and lung cancer. This in situ method offers single cell mutation detection for diagnostics and holds great promise as a tool to investigate the role of oncogenic mutations in complex tumor tissues.

Keyword [en]
mutation, KRAS, padlock probes, rolling circle amplification, in situ, FFPE, touch imprints, cancer, diagnostics
National Category
Medical Genetics Medical Genetics Cancer and Oncology
Research subject
Molecular Medicine; Oncology
Identifiers
URN: urn:nbn:se:uu:diva-149822OAI: oai:DiVA.org:uu-149822DiVA: diva2:405867
Available from: 2011-03-24 Created: 2011-03-24 Last updated: 2011-05-05
In thesis
1. Genotyping and Mutation Detection In Situ: Development and application of single-molecule techniques
Open this publication in new window or tab >>Genotyping and Mutation Detection In Situ: Development and application of single-molecule techniques
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human body is composed of trillions of cells closely working together to maintain a functional organism. Every cell is unique in molecular composition and can acquire genetic variations that might cause it to turn pathological. It is essential to develop improved tools to better understand the development of normal and disease tissue, ideally enabling single-cell expression studies in preserved context of complex tissue with single-nucleotide resolution. This thesis presents the development and application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The described technique utilizes padlock probes and target-primed rolling circle amplification and is highly suitable for sensitive in situ analysis.

First, a new strategy for directed cleavage of single stranded DNA was investigated, e.g. nucleic acid targets with extended 3´ ends, for successful initiation of rolling circle amplification. The presented cleavage strategy is simple and applicable for subsequent enzymatic reactions, e.g. ligation and polymerization. Specific cleavage of long target overhangs was demonstrated in synthetic oligonucleotides and in genomic DNA and the detection efficiency was substantially increased.

For multiplex detection and genotyping of individual transcripts in single cells, a new in situ method was developed. The technique showed a satisfactorily detection efficiency and was later applied as a general mutation analysis tool for detection of KRAS point mutations in complex tumor tissue sections, e.g. formalin-fixed, paraffin-embedded tumor tissues and cytologic tumor imprints. Mutation status was assessed in patient samples by in situ padlock probe detection and results were confirmed by DNA-sequencing.  Finally, the method was adapted for simultaneous detection of individual mRNA molecules and endogenous protein modifications in single cells using padlock probes and in situ PLA. This assay will be useful for gene expression analysis and exploration of new drugs with vague effector sites.

To our knowledge, no other technique exists today that offers in situ transcript detection with single-nucleotide resolution in heterogeneous tissues. The method will especially be suitable for discrimination of highly similar transcripts, e.g. splice variants, SNPs and point mutations, within gene expression studies and for cancer diagnostics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 656
Keyword
padlock probes, in situ, rolling circle amplification, mRNA, genotyping, mutation detection, cancer, tissue sections, diagnostics, single-molecule, single-cell, microscopy
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-149776 (URN)978-91-554-8034-9 (ISBN)
Public defence
2011-05-06, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-04-14 Created: 2011-03-23 Last updated: 2011-05-05Bibliographically approved

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