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From Tissue to Mutations: Genetic Profiling of Colorectal Cancer
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Comprehensive characterisation of the mutational landscapes of solid tumours is a multistep process involving the collection of suitable samples, the extraction of nucleic acids and the preparation of these materials for mutational analyses. In this thesis, I aimed to develop a streamlined process for the analysis of colorectal cancer (CRC) patient samples in order to identify novel mutations that hallmark the development of advanced disease.

Papers I and II outline a technique for serial extraction of nucleic acids from frozen tissue that we developed and subsequently implemented on a robotic platform to enable high-throughput processing. The extracted nucleic acids were validated in downstream processes relevant for genetic analyses, including traditional Sanger and next generation sequencing  techniques.

In Paper III, we developed a genotyping method based on multiplex ligation-dependent genome amplification. The method was designed such that InDel polymorphisms of between 30 and 70 % prevalence in a European population were selected and amplified in a multiplex PCR assay. DNA from 24 patient-matched colorectal tumour and normal tissues was genotyped and paired with a high match probability.

In Paper IV, we performed targeted resequencing of 107 primary CRCs, of which approximately half developed metastatic disease or had distant metastases at the time of diagnosis. We chose to analyse 676 genes based on their involvement in key signalling pathways in CRC. We found an enrichment of mutations in the Eph receptor tyrosine kinase gene family in metastatic patients, indicating a potential role for these genes in CRC metastasis.

This thesis outlines a series of procedures that can be employed in a high-throughput setting for the analysis of solid tumours. We applied these methods to the analysis of colorectal tumours and propose a link between novel somatic mutations and metastatic disease.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1028
Keyword [en]
Nucleic acid extraction, genotyping, targeted sequencing, mutation analysis, colorectal cancer, metastatic disease
National Category
Medical Genetics Cancer and Oncology
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-231508ISBN: 978-91-554-9042-3 (print)OAI: oai:DiVA.org:uu-231508DiVA: diva2:748676
Public defence
2014-11-07, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-10-16 Created: 2014-09-08 Last updated: 2015-01-23
List of papers
1. Efficient and scalable serial extraction of DNA and RNA from frozen tissue samples
Open this publication in new window or tab >>Efficient and scalable serial extraction of DNA and RNA from frozen tissue samples
2010 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 47, no 1, 547-549 p.Article in journal (Refereed) Published
Abstract [en]

Advances in cancer genomics have created a demand for scalable sample processing. We here present a process for serial extraction of nucleic acids from the same frozen tissue sample based on magnetic silica particles. The process is automation friendly with high recoveries of pure DNA and RNA suitable for analysis.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-140159 (URN)10.1039/c0cc02248a (DOI)000285068300136 ()21103565 (PubMedID)
Available from: 2011-01-04 Created: 2011-01-04 Last updated: 2017-12-11Bibliographically approved
2. Automated serial extraction of DNA and RNA from biobanked tissue specimens
Open this publication in new window or tab >>Automated serial extraction of DNA and RNA from biobanked tissue specimens
2013 (English)In: BMC Biotechnology, ISSN 1472-6750, E-ISSN 1472-6750, Vol. 13, 66- p.Article in journal (Refereed) Published
Abstract [en]

Background: With increasing biobanking of biological samples, methods for large scale extraction of nucleic acids are in demand. The lack of such techniques designed for extraction from tissues results in a bottleneck in downstream genetic analyses, particularly in the field of cancer research. We have developed an automated procedure for tissue homogenization and extraction of DNA and RNA into separate fractions from the same frozen tissue specimen. A purpose developed magnetic bead based technology to serially extract both DNA and RNA from tissues was automated on a Tecan Freedom Evo robotic workstation. Results: 864 fresh-frozen human normal and tumor tissue samples from breast and colon were serially extracted in batches of 96 samples. Yields and quality of DNA and RNA were determined. The DNA was evaluated in several downstream analyses, and the stability of RNA was determined after 9 months of storage. The extracted DNA performed consistently well in processes including PCR-based STR analysis, HaloPlex selection and deep sequencing on an Illumina platform, and gene copy number analysis using microarrays. The RNA has performed well in RT-PCR analyses and maintains integrity upon storage. Conclusions: The technology described here enables the processing of many tissue samples simultaneously with a high quality product and a time and cost reduction for the user. This reduces the sample preparation bottleneck in cancer research. The open automation format also enables integration with upstream and downstream devices for automated sample quantitation or storage.

Keyword
Nucleic acid extraction, Cancer, Open automation, Sample preparation, Tissue biobanking
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-207527 (URN)10.1186/1472-6750-13-66 (DOI)000323342100001 ()
Available from: 2013-09-17 Created: 2013-09-16 Last updated: 2017-12-06Bibliographically approved
3. Automated Genotyping of Biobank Samples by Multiplex Amplification of Insertion/Deletion Polymorphisms
Open this publication in new window or tab >>Automated Genotyping of Biobank Samples by Multiplex Amplification of Insertion/Deletion Polymorphisms
Show others...
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 12, e52750- p.Article in journal (Refereed) Published
Abstract [en]

The genomic revolution in oncology will entail mutational analyses of vast numbers of patient-matched tumor and normal tissue samples. This has meant an increased risk of patient sample mix up due to manual handling. Therefore, scalable genotyping and sample identification procedures are essential to pathology biobanks. We have developed an efficient alternative to traditional genotyping methods suited for automated analysis. By targeting 53 prevalent deletions and insertions found in human populations with fluorescent multiplex ligation dependent genome amplification, followed by separation in a capillary sequencer, a peak spectrum is obtained that can be automatically analyzed. 24 tumor-normal patient samples were successfully matched using this method. The potential use of the developed assay for forensic applications is discussed.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-193620 (URN)10.1371/journal.pone.0052750 (DOI)000312829100077 ()
Available from: 2013-02-06 Created: 2013-02-05 Last updated: 2017-12-06Bibliographically approved
4. Somatic Eph receptor mutations in primary colorectal cancers are associated with metastasis
Open this publication in new window or tab >>Somatic Eph receptor mutations in primary colorectal cancers are associated with metastasis
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Medical Genetics
Research subject
Medical Genetics; Molecular Genetics
Identifiers
urn:nbn:se:uu:diva-231503 (URN)
Available from: 2014-09-08 Created: 2014-09-08 Last updated: 2015-01-23

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