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The use of formalin fixed paraffin embedded tissue and global gene expression profiling for increased understanding of squamous cell carcinoma of the tongue
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Head and neck cancer is the 6th most common malignancy worldwide, with tumours of the tongue being one of the most prevalent sites. Despite advances in surgery and radiotherapy, the five-year survival has not changed during the last decades and remains at approximately 50%. Identification of novel biomarkers for more personalized treatment is important for increasing survival in these patients. One of the most commonly used methods in the search for new biomarkers is microarray analysis. A substantial limitation with this technique is the requirement for fresh frozen samples from which high quality RNA can be extracted. This becomes particularly problematic when attempting to discover differences associated with individual sub-types or rare cancers. Recent developments, including the DASL microarray platform, have provided the possibility of analysing RNA of poorer quality from formalin fixed paraffin embedded (FFPE) samples. FFPE is the standard way of preserving tissue from patients and millions of samples are stored around the world. In this thesis we have evaluated the use of FFPE samples and global gene expression profiling for increasing basic knowledge in a subgroup of oral cancer patients with tumours of the tongue.

As confirmation of microarray results using qPCR is of outmost importance for conclusive data evaluation, we first aimed at finding a housekeeping gene stably expressed across malignant and non-malignant FFPE oral tissue. TUBA6, which belongs to the tubulin family was detected as being the most stable out of eight possible genes and was thus used for qPCR normalization throughout the following studies.

We have performed three separate microarray experiments. Initially only a focused DASL array covering 502 cancer related genes was available and we used it to analyze a smaller cohort of patients and controls (n=36). A similar cohort (n=29) was also analyzed for expression of 836 micoRNAs. In 2009 a whole genome DASL array was launched, covering over 20,000 genes, and all tongue tumour samples available between 1997 and 2010 (n=87) were analysed using this array.

Similar to other research groups we observed very high replicate reproducibility using both DASL arrays. When using the microRNA array and the whole genome DASL array an effect of sample quality on the detected expression level of individual genes was noticed. While the expression of some genes severely decreased with a decrease in sample quality others were not changed. This will impair normalization, leading to a residual non-biological variation within the data. Based on our findings we have presented some recommendations for minimizing the effect of sample quality and maximizing the level of biologically relevant information obtained from these experiments, e.g. ensuring that samples in groups to be compared are of the same quality range. For the microRNA data we also introduced an additional normalization step to the standard normalizations. We could show that lists of differentially expressed genes generated when taking these precautions were enriched for genes involved in cancer related processes and contained for tongue carcinoma previously identified changes. A number of differentially expressed genes, novel for tongue carcinoma, were also confirmed in high quality fresh frozen samples, including BCL2A1 (apoptosis), CXCL10 (immune response), SLC2A6 (energy transport) and miR-424 (angiogenesis).

In conclusion microarrays can be used to analyze FFPE samples but should be performed with care. Standard normalization methods will not remove the variation introduced by samples being of different quality, leading to spurious results. Taking a few precautions, however, led to the identification of differentially expressed genes relevant in tumour development and maintenance. The recommendations we make can facilitate design of future studies using FFPE samples. The genes we identified as being differentially expressed in tumour tissue now need to be further evaluated for their potential as biomarkers in tongue carcinoma.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2012. , 45 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1495
Keyword [en]
SCCHN, tongue, microarray, DASL, FFPE, archival, mRNA, miRNA
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Pathology
Identifiers
URN: urn:nbn:se:umu:diva-54005ISBN: 978-91-7459-413-3 (print)OAI: oai:DiVA.org:umu-54005DiVA: diva2:514881
Public defence
2012-05-04, Betula, byggnad 6M, Norrlands Universitetsjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-04-13 Created: 2012-04-11 Last updated: 2012-07-17Bibliographically approved
List of papers
1. Tubulin-α-6-chain is a stably expressed reference gene in archival samples of normal oral tissue and squamous cell carcinoma of the head and neck
Open this publication in new window or tab >>Tubulin-α-6-chain is a stably expressed reference gene in archival samples of normal oral tissue and squamous cell carcinoma of the head and neck
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2010 (English)In: Experimental and Therapeutic Medicine, ISSN 1792-0981, Vol. 1, no 3, 419-423 p.Article in journal (Refereed) Published
Abstract [en]

One of the most critical factors in gene expression studies using quantitative real-time PCR is the choice of reference gene. Many of the commonly used reference genes have been shown to vary during a number of biological processes as well as between tissues. It is therefore important to always verify the stability of the gene of choice for all new tissues and experimental conditions. Here, we used two publicly available computer software packages (GeNorm and NormFinder) to investigate the stability of eight potential reference genes in formalin-fixed paraffin-embedded (FFPE) samples from normal oral tissue of different origin as well as from oral squamous cell carcinomas. Both programs found the tubulin α-6 chain (TUBA6) and ribosomal protein S13 (RPS13) to have the most stable expression between malignant and non-malignant tissue. NormFinder also found TUBA6 to be the most stable gene when samples were grouped according to tissue origin. FFPE samples constitute a large research resource, which considerably increases the number of samples available for analysis, leading to more reliable conclusions. Verification of a proper reference gene in oral FFPE tissue is therefore of great importance for future studies.

Keyword
oral squamous cell carcinoma, reference gene, tubulin a-6 chain, quantitive real-time PCR
National Category
Cancer and Oncology
Research subject
Odontology
Identifiers
urn:nbn:se:umu:diva-40751 (URN)10.3892/etm_00000065 (DOI)000284799800003 ()
Available from: 2011-03-09 Created: 2011-03-09 Last updated: 2012-04-13Bibliographically approved
2. Gene expression profiling of archival tongue squamous cell carcinomas provides sub-classification based on DNA repair genes
Open this publication in new window or tab >>Gene expression profiling of archival tongue squamous cell carcinomas provides sub-classification based on DNA repair genes
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2009 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 35, no 6, 1321-1330 p.Article in journal (Refereed) Published
Abstract [en]

A subgroup of patients with squamous cell carcinoma of the head and neck (SCCHN) comprise young persons under the age of 40, who have not been heavily exposed to the classical risk factors, smoking and alcohol. The number of SCCHN in young adults, particularly tongue tumours, is increasing in several parts of the world. Here we employed a novel gene expression array methodology specifically developed for analysis of degraded RNA and investigated the expression of 502 cancer-related genes in archival paraffin-embedded SCCHN of the tongue from young (< or =40) and elderly patients (> or =50). Genes detected as de-regulated in tumours compared to non-malignant controls were in concordance with results from earlier studies of fresh frozen material. No genes were detected as significantly differentially expressed between young and old patients suggesting that the overall pathobiology of SCCHN is similar in young and old. Unsupervised clustering divided tumours into three groups, irrespective of age, where several differentially expressed DNA repair genes were a prominent separation factor. High levels of DNA repair genes associated with impaired therapeutic response to radiation, suggesting that DNA repair genes play a role in clinical outcome after radiotherapy.

Keyword
microaray; formalin-fixed paraffin-embedded; squamous cell carcinoma of the head and neck; tongue; young adults
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-30367 (URN)10.3892/ijo_00000450 (DOI)19885555 (PubMedID)
Available from: 2009-12-18 Created: 2009-12-18 Last updated: 2012-04-13Bibliographically approved
3. Transcriptional profiling of formalin fixed paraffin embedded tissue: pitfalls and recommendations for identifying biologically relevant changes
Open this publication in new window or tab >>Transcriptional profiling of formalin fixed paraffin embedded tissue: pitfalls and recommendations for identifying biologically relevant changes
2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 4, e35276- p.Article in journal (Refereed) Published
Abstract [en]

Expression profiling techniques have been used to study the biology of many types of cancer but have been limited to some extent by the requirement for collection of fresh tissue. In contrast, formalin fixed paraffin embedded (FFPE) samples are widely available and represent a vast resource of potential material. The techniques used to handle the degraded and modified RNA from these samples are relatively new and all the pitfalls and limitations of this material for whole genome expression profiling are not yet clarified. Here, we analyzed 70 FFPE tongue carcinoma samples and 17 controls using the whole genome DASL array covering nearly 21000 genes. We identified that sample age is related to quality of extracted RNA and that sample quality influences apparent expression levels in a non-random manner related to gene probe sequence, leading to spurious results. However, by removing sub-standard samples and analysing only those 28 cancers and 15 controls that had similar quality we were able to generate a list of 934 genes significantly altered in tongue cancer compared to control samples of tongue. This list contained previously identified changes and was enriched for genes involved in many cancer-related processes such as tissue remodelling, inflammation, differentiation and apoptosis. Four novel genes of potential importance in tongue cancer development and maintenance, SH3BGL2, SLC2A6, SLC16A3 and CXCL10, were independently confirmed, validating our data. Hence, gene expression profiling can be performed usefully on archival material if appropriate quality assurance steps are taken to ensure sample consistency and we present some recommendations for the use of FFPE material based on our findings.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-57314 (URN)10.1371/journal.pone.0035276 (DOI)000305347400040 ()22530001 (PubMedID)
Available from: 2012-07-11 Created: 2012-07-11 Last updated: 2017-12-07Bibliographically approved
4. miRNA analysis of formalin-fixed squamous cell carcinomas of the tongue is affected by age of the samples
Open this publication in new window or tab >>miRNA analysis of formalin-fixed squamous cell carcinomas of the tongue is affected by age of the samples
Show others...
2011 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 38, no 1, 61-69 p.Article in journal (Refereed) Published
Abstract [en]

Global miRNA expression arrays were used for analysis of 836 miRNAs in formalin-fixed paraffin-embedded samples from 21 tongue cancer patients and 8 controls. Samples had been stored for one to eleven years. Results separated tumour samples from controls, however, the largest variation was correlated to sample storage time, detectable already after one year. With the use of a linear regression model we could adjust for the storage-dependent effect, leading to the identification of 54 differentially expressed miRNAs in tongue cancer, compared to 16 when using standard normalization, including up-regulation of a novel miRNA, miR-424.

Place, publisher, year, edition, pages
Spandidos Publ. Ltd, 2011
Keyword
miRNA, FFPE, tongue carcinoma, storage, normalization
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-38654 (URN)10.3892/ijo_00000824 (DOI)000286293000008 ()21109926 (PubMedID)
Available from: 2010-12-20 Created: 2010-12-20 Last updated: 2017-12-11Bibliographically approved

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