| 1. |
- Danielsson, Karin, 1965-, et al.
(författare)
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Autoantibodies and decreased expression of the transcription factor ELF-3 together with increased chemokine pathways support an autoimmune phenotype and altered differentiation in lichen planus located in oral mucosa
- 2013
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Ingår i: Journal of the European Academy of Dermatology and Venereology. - : Wiley-Blackwell. - 0926-9959 .- 1468-3083. ; 27:11, s. 1410-1416
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Tidskriftsartikel (refereegranskat)abstract
- Background The pathogenesis of oral lichen planus (OLP), a chronic inflammatory disease, is not fully understood. It is known that OLP has autoimmune features, and it is suggested to be an autoimmune disease. ELF-3 is involved in differentiation of keratinocytes and deregulated in different tumours and inflammatory diseases. CXCR-3 and its ligands CXCL-10 and CXCL-11 are increased in autoimmune diseases and linked to Th-1 immune response. Objectives To analyse and compare expression of ELF-3, CXCR-3, CXCL-10 and CXCL-11 in OLP lesions and controls in whole and microdissected epithelium. Methods Tissue biopsies from 20 patients clinically and histologically diagnosed with OLP and 20 healthy controls were studied using whole tissues or microdissected epithelium. By the use of qRT-PCR, mRNA levels of ELF-3, CXCR-3, CXCL-10 and CXCL-11 were studied. Western blot was used for analysis of ELF-3 protein expression. Sera from 19 OLP patients and 20 controls were analysed with ELISA in search for autoantibodies. Results The upregulation of CXCR-3, CXCL-10 and CXCL-11 found in OLP is similar to previous findings showing an autoimmune phenotype in lichen planus (LP) and lichen sclerosus. Decreased expression of the differentiation-related transcription factor ELF-3 was also seen in OLP lesions, and we further demonstrate presence of circulating autoantibodies against the ELF-3 protein in sera from 3 of 19 (16%) LP patients tested. Conclusions On the basis of these findings, we confirm that OLP shows features of an autoimmune disease and suggest deregulated differentiation of keratinocytes to be one of the causes of the disease phenotype.
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| 2. |
- Fahlén, Jessica, 1973-, et al.
(författare)
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MicroRNA-microarray data analysis in the precence of FFPE storage time effects
- 2010
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background: The standard method for preserving patient samples for diagnostic purposes is fixation in formalin followed by embedding in paraffin (FFPE). The use of FFPE blocks makes it possible to include a large number of patients in the experimental studies since millions of FFPE blocks are stored around the world. However, FFPE storage can cause degradation and modifications of nucleic acids. In order to draw reliable biological conclusions it is therefore important to know what effect FFPE-storage have on the tissues and to have procedures that normalize this effect. In this paper, we study the effect that FFPE-storage has on microRNA-microarray data from tongue-cancer patients and propose a novel procedure for normalizing the bias introduced by FFPE-storage.Results: MicroRNA-microarray data from 21 tongue-cancer patients and 8 control patients were used. The samples were stored in FFPE blocks and had been in storage for up to 11 years. The data contained a large amount of biological relevant variation, yet the largest variation was due to the samples storage times. The storage effect was shown to be significant and some results suggested that it may be causal. Moreover, the microRNAs were unequally affected by storage and this could partially be explained by sequence characteristics. The novel normalization procedure was shown to have a large impact in the analysis ability to identify differentially expressed microRNAs between young and old cancer patients as well as between cancer and control patients. The p-values for the top microRNAs candidates were much lower for the proposed novel normalization compared to a standard normalization procedure which suggested that the novel normalization made the analysis more efficient.Conclusions: MicroRNA-microarray data can be seriously affected by FFPE-storage and the introduced variation cannot be removed by standard normalizations. The proposed normalization removes the bias introduced by FFPE-storage and gives higher sensitivity than the standard normalization.
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| 3. |
- Matilda, Rentoft, 1981-
(författare)
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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
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
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| 5. |
- Rentoft, Matilda, et al.
(författare)
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Expression of CXCL10 is associated with response to radiotherapy and overall survival in squamous cell carcinoma of the tongue
- 2014
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Ingår i: Tumor Biology. - : Springer Science and Business Media LLC. - 1010-4283 .- 1423-0380. ; 35:5, s. 4191-4198
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Tidskriftsartikel (refereegranskat)abstract
- Five-year survival for patients with oral cancer has been disappointingly stable during the last decades, creating a demand for new biomarkers and treatment targets. Lately, much focus has been set on immunomodulation as a possible treatment or an adjuvant increasing sensitivity to conventional treatments. The objective of this study was to evaluate the prognostic importance of response to radiotherapy in tongue carcinoma patients as well as the expression of the CXC-chemokines in correlation to radiation response in the same group of tumours. Thirty-eight patients with tongue carcinoma that had received radiotherapy followed by surgery were included. The prognostic impact of pathological response to radiotherapy, N-status, T-stage, age and gender was evaluated using Cox's regression models, Kaplan-Meier survival curves and chi-square test. The expression of 23 CXC-chemokine ligands and their receptors were evaluated in all patients using microarray and qPCR and correlated with response to treatment using logistic regression. Pathological response to radiotherapy was independently associated to overall survival with a 2-year survival probability of 81% for patients showing a complete pathological response, while patients with a non-complete response only had a probability of 42% to survive for 2 years (p = 0.016). The expression of one CXC-chemokine, CXCL10, was significantly associated with response to radiotherapy and the group of patients with the highest CXCL10 expression responded, especially poorly (p = 0.01). CXCL10 is a potential marker for response to radiotherapy and overall survival in patients with squamous cell carcinoma of the tongue.
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| 6. |
- Rentoft, Matilda, et al.
(författare)
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miRNA analysis of formalin-fixed squamous cell carcinomas of the tongue is affected by age of the samples
- 2011
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Ingår i: International Journal of Oncology. - : Spandidos Publ. Ltd. - 1019-6439 .- 1791-2423. ; 38:1, s. 61-69
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
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| 8. |
- Rentoft, Matilda, et al.
(författare)
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Transcriptional profiling of formalin fixed paraffin embedded tissue : pitfalls and recommendations for identifying biologically relevant changes
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:4, s. e35276-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Rentoft, Matilda, et al.
(författare)
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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
- 2010
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Ingår i: Experimental and Therapeutic Medicine. - : Spandidos Publications. - 1792-0981 .- 1792-1015. ; 1:3, s. 419-423
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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