PubMed Health⌕ Search

Biomedical subjects

Torben F Ørntoft

Publications and source records attributed to Torben F Ørntoft.

18 recordsLinked to original sources
PubMed2006

Validation of the use of DNA pools and primer extension in association studies of sporadic colorectal cancer for selection of candidate SNPs.

Colorectal cancer (CRC) is a multifactorial disease that involves both lifestyle and genetic factors. To identify single nucleotide polymorphisms (SNPs) associated with sporadic CRC, we used pooled DNA samples representing 230 cases with sporadic CRC and 540 controls. The allele frequency of the SNPs was estimated in the two pools using a genotyping method based on primer extension and capillary electrophoresis (CE). The sensitivity of the method was high, which permitted the detection of an odds ratio (OR) of 1.5. Validation of the method showed that it is robust, linear, sensitive, and reproducible. Of the 224 SNPs investigated, 20 potential candidates associated with CRC were identified, including IL6 -174G>C (g.22062318G>C), XRCC1 c.685 C>T (p.Arg194Trp), PPARGC1A g.92945042C>T (3'UTR 96516), GSTP1 c.342A>C (p.Ile105Val), GSTM1 c.573C>G (p.Lys173Asn), and SULT1A1 g.19934792G>A (p.Arg213His). All were borderline significant, and none were significant at the 5% level. A high number of the SNPs (40%) were not polymorphic in our population. We conclude that instead of looking for single risk factors, investigators should examine individual combinations of potential risk factors to clarify the genetic predisposition to CRC.

Aged↗
PubMed2006

Folate deficiency induced hyperhomocysteinemia changes the expression of thrombosis-related genes.

Hyperhomocysteinemia (HH) is an independent risk factor for thrombosis although the precise pathogenesis is still unresolved. Previous studies have demonstrated that HH changes whole blood coagulation by increasing the velocity, increasing the firmness of the formed clot, and by prolonging the initiation phase of the coagulation. With the aim of elucidating the genetic pathogenesis which might be responsible for the changes in whole blood coagulation, we applied oligo-array technology to RNA from buffycoat-cells comparing animals suffering from hyperhomocysteinemia (42 micromol/l) with controls (6 micromol/l). Data mining identified a number of relevant genes, and the expression pattern was validated by real time reverse transcriptase-polymerase chain reaction. An upregulation of integrin beta-3, Rap 1b, glycoprotein V, platelet-endothelial cell adhesion molecule-1 (PECAM-1) and von Willebrand factor (vWF) led us to deduce increased platelet activation/aggregation. Coagulation factor XIIIa was upregulated and may contribute in increasing the firmness of the formed clot. Impaired fibrinolysis was anticipated, since an upregulation of plasminogen activator inhibitor-1 (PAI-1) and a downregulation of tissue-type plasminogen activator (t-PA) were detected. Reduced spontaneous contact activation was anticipated due to a downregulation of the kallikrein gene. Upregulation of selectins may contribute to increased tethering and rolling of leukocytes. In conclusion, folate deficiency induced hyperhomocysteinemia changes in the gene expression of buffy coat cells which was characterized by increased platelet activation, impaired fibrinolysis and a reduced contact activation of the coagulation. These changes may contribute to explain the increased risk of thrombosis seen in hyperhomocysteinemia individuals. This pattern of the hyperhomocysteinemia-affected genes may represent a reference for further studies at the protein level to define the folate depletion effects in blood cells.

Animals↗
PubMed2006

Recent developments in molecular profiling of bladder cancer.

PURPOSE OF REVIEW: Profiling bladder tumors using high-density microarrays has recently become possible. We review recent reports on the use of profiling for the prediction of various clinical courses in bladder cancer. We also stress the methods and materials needed to translate such molecular profiles into clinically useful tests. RECENT FINDINGS: Using gene expression microarrays, it has been possible to identify signatures that predict various properties of bladder cancer such as stage, grade, progression, and likelihood of metastases. Using arrays for genomic instability, similar properties have been examined, but the effectiveness seems to be less than that of gene expression. Some of genes identified also work as predictors using immunohistochemistry. SUMMARY: The signatures or molecular profiles for some of these arrays are now being tested in multicenter studies with the purpose of introducing these into the clinic, for planning of follow-up and treatment selection.

Biomarkers, Tumor↗
PubMed2005

Construction and validation of the APOCHIP, a spotted oligo-microarray for the study of beta-cell apoptosis.

BACKGROUND: Type 1 diabetes mellitus (T1DM) is a autoimmune disease caused by a long-term negative balance between immune-mediated beta-cell damage and beta-cell repair/regeneration. Following immune-mediated damage the beta-cell fate depends on several genes up- or down-regulated in parallel and/or sequentially. Based on the information obtained by the analysis of several microarray experiments of beta-cells exposed to pro-apoptotic conditions (e.g. double stranded RNA (dsRNA) and cytokines), we have developed a spotted rat oligonucleotide microarray, the APOCHIP, containing 60-mer probes for 574 genes selected for the study of beta-cell apoptosis. RESULTS: The APOCHIP was validated by a combination of approaches. First we performed an internal validation of the spotted probes based on a weighted linear regression model using dilution series experiments. Second we profiled expression measurements in ten dissimilar rat RNA samples for 515 genes that were represented on both the spotted oligonucleotide collection and on the in situ-synthesized 25-mer arrays (Affymetrix GeneChips). Internal validation showed that most of the spotted probes displayed a pattern of reaction close to that predicted by the model. By using simple rules for comparison of data between platforms we found strong correlations (rmedian= 0.84) between relative gene expression measurements made with spotted probes and in situ-synthesized 25-mer probe sets. CONCLUSION: In conclusion our data suggest that there is a high reproducibility of the APOCHIP in terms of technical replication and that relative gene expression measurements obtained with the APOCHIP compare well to the Affymetrix GeneChip. The APOCHIP is available to the scientific community and is a useful tool to study the molecular mechanisms regulating beta-cell apoptosis.

Algorithms↗
PubMed2005

Increase in amphiregulin and epiregulin in prostate cancer xenograft after androgen deprivation-impact of specific HER1 inhibition.

BACKGROUND: We investigated the expression of the epidermal growth factor (EGF) network before and after castration in the prostate cancer xenograft CWR22 implanted in nude mice, and examined the effects of gefitinib (Iresssa, ZD1839), a new drug directed towards the EGF tyrosine kinase receptor (HER1) of the EGF network. METHODS: CWR22 prostate cancer xenografts were propagated in immunodeficient male mice. The expression of the growth factors and receptors in the EGF network was examined by real-time PCR analysis and ELISA at 0, 7, 14, and 30 days after castration, and the tumor growth was examined after treatment with gefitinib or placebo. RESULTS: A fraction of growth factors showed a steady increase in the mRNA expression reaching between fourfold and eightfold 30 days after castration, including amphiregulin (P < 0.005) and epiregulin (P < 0.001). ELISA for amphiregulin showed a fivefold increase 30 days after castration. Tumor bearing mice were castrated and treated with or without the HER1 tyrosine kinase inhibitor gefitinib. Tumor involution was significantly increased by castration plus gefitinib as compared to castration alone. CONCLUSIONS: Castration leads to adaptive increase in the concentrations of a subset of growth factors from the EGF network in the androgen sensitive CWR22 prostate cancer xenograft. Specific inhibition of the HER1 tyrosine kinase receptor significantly increases the tumor involution, and suggests that HER1 targeted drugs could be of therapeutic relevance in the treatment of advanced prostate cancer.

Amphiregulin↗
PubMed2004

Aberrations of the Chk2 tumour suppressor in advanced urinary bladder cancer.

Checkpoint kinase 2 (Chk2) is a tumour suppressor and signal transducer in genome integrity checkpoints that coordinate cell-cycle progression with DNA repair or cell death in response to DNA damage. Defects of Chk2 occur in subsets of diverse sporadic malignancies and predispose to several types of hereditary carcinomas. However, the status of Chk2 in tumours of the urinary bladder remains unknown. Here, we report that among 58 advanced (grade T2-T4) human bladder carcinomas, immunohistochemical analysis revealed tumour-specific reduction or lack of Chk2 protein in 6 (10.3%) cases. Genetic analysis of the latter subset showed that a Chk2-negative carcinoma #668 harboured a truncating mutation 1100delC, in one Chk2 allele and loss of the corresponding second allele. The 1100delC mutation was also found in the germ line of this patient. Sequencing of TP53 in tumour #668 identified two missense mutations. Furthermore, the vast majority of the tumours showed 'unscheduled' activatory phosphorylation on Thr68 of Chk2 in the absence of any DNA-damaging treatment. Our results indicate that the otherwise dormant DNA damage signal transducer Chk2 is aberrantly and constitutively activated in invasive urinary bladder carcinomas, and that such likely proapoptotic checkpoint signalling can be disabled by inactivation of Chk2 and/or p53 tumour suppressors in subsets of these tumours.

Carcinoma↗
PubMed2004

Gene expression in the urinary bladder: a common carcinoma in situ gene expression signature exists disregarding histopathological classification.

The presence of carcinoma in situ (CIS) lesions in the urinary bladder is associated with a high risk of disease progression to a muscle invasive stage. In this study, we used microarray expression profiling to examine the gene expression patterns in superficial transitional cell carcinoma (sTCC) with surrounding CIS (13 patients), without surrounding CIS lesions (15 patients), and in muscle invasive carcinomas (mTCC; 13 patients). Hierarchical cluster analysis separated the sTCC samples according to the presence or absence of CIS in the surrounding urothelium. We identified a few gene clusters that contained genes with similar expression levels in transitional cell carcinoma (TCC) with surrounding CIS and invasive TCC. However, no close relationship between TCC with adjacent CIS and invasive TCC was observed using hierarchical cluster analysis. Expression profiling of a series of biopsies from normal urothelium and urothelium with CIS lesions from the same urinary bladder revealed that the gene expression found in sTCC with surrounding CIS is found also in CIS biopsies as well as in histologically normal samples adjacent to the CIS lesions. Furthermore, we also identified similar gene expression changes in mTCC samples. We used a supervised learning approach to build a 16-gene molecular CIS classifier. The classifier was able to classify sTCC samples according to the presence or absence of surrounding CIS with a high accuracy. This study demonstrates that a CIS gene expression signature is present not only in CIS biopsies but also in sTCC, mTCC, and, remarkably, in histologically normal urothelium from bladders with CIS. Identification of this expression signature could provide guidance for the selection of therapy and follow-up regimen in patients with early stage bladder cancer.

Biopsy↗
PubMed2004

High-density oligonucleotide array analysis reveals extensive differences between freshly isolated blood and hepatic natural killer cells.

Hepatic NK cells are more cytotoxic than blood NK cells against tumor cells. To understand the basis of this difference in cytotoxicity we analyzed RNA derived from freshly isolated rat blood and hepatic NK cells [high-density (HD) and low-density subpopulations] by high-density oligonucleotide arrays (Affymetrix), containing about 9,000 genes and expressed sequence tags. IL-2-treated blood NK (A-NK) cells and IL-2-treated hepatic HD cells were used as a reference of NK cell activation. About 150 genes and expressed sequence tags were differentially expressed between hepatic and blood NK cells. Surprisingly, more than half of the increased expressed genes in hepatic NK cells were not increased in A-NK cells. Differentially expressed genes like the stem cell factor receptor c-kit and the chemokine receptor CCR5 can contribute to the homing and differentiation of hepatic NK cells in the liver sinusoids. Several of the differentially expressed genes can possibly contribute to the enhanced cytotoxic activity of hepatic NK cells: cell membrane receptors like NKG2D, NKG2C, CD94, ecto-ATPase; signaling molecules like phosphatidylinositol 3-kinase; granule-associated effector molecules like granzymes and defensin NP3. Moreover, it appears that redirection of cytotoxic granules and increase in intracellular Ca2+ are convergence points of several of these genes.

Animals↗
PubMed2003

Classification of Dukes' B and C colorectal cancers using expression arrays.

PURPOSE: Colorectal cancer is one of the most common malignancies. Substaging of the cancer is of importance not only to prognosis but also to treatment. Classification of substages based on DNA microarray technology is currently the most promising approach. We therefore investigated if gene expression microarrays could be used to classify colorectal tumors. METHODS: We used the Affymetrix oligonucleotide arrays to analyze the expression of more than 5,000 genes in samples from the sigmoid and upper rectum of the left colon. Five samples were from normal mucosa and five samples from each of the Dukes' stages A, B, C, and D. Expression data were filtered based on either covariance or a selection of the most significantly varying genes between tumor stages. RESULTS: A nearest neighbor classifier was used to classify normal, and Dukes' B and C samples with less than 20% error, whereas Dukes' A and D could not be classified correctly. A number of interesting gene clusters showed a discriminating difference between Dukes' B and C samples. These included mitochondrial genes, stromal remodeling genes, and genes related to cell adhesion. CONCLUSION: Molecular classification based on gene expression of one of the most common malignancies, colorectal cancer, now seems to be within reach. The data indicates that it is possible at least to classify Dukes' B and C colorectal tumors with microarrays.

Adenocarcinoma↗
PubMed2003

Genome-wide allelotyping of 104 Finnish colorectal cancers reveals an excess of allelic imbalance in chromosome 20q in familial cases.

We have allelotyped a series of 104 Finnish colorectal cancers (CRCs) using 372 polymorphic markers spaced, on average, at 10 cM intervals, and have made a comparison of the differences in the frequency of allelic imbalance (AI) between familial and sporadic cases. Differences in the frequency of allelic imbalance (loss of heterozygosity or amplification) at a number of loci were detected and these were evaluated through analysis of additional series of cancers using specific markers. The most consistent difference was observed at chromosome 20q13.1-13.3 characterized by a two fold difference between familial and nonfamilial disease in a total of 99 familial and 186 sporadic Finnish cases. This difference was not observed in a UK set of 67 familial and 96 sporadic CRCs. The genome-wide effort resulted in a large data set giving clues to the location of putative CRC predisposition genes in the genome. The approach provides an alternative strategy for detecting cancer predisposition genes solely reliant on the molecular analysis of single cases obviating the requirement to collect multiple samples from families.

Adult↗
PubMed2003

[DNA microarrays (DNA chips) used in molecular medical research].

Microchip technology and mapping of the human genome has led to the invention of DNA-micro-array technology. In that technology thousands of genes are analysed in parallel on a small glass surface. The very large data material that is generated requires bioinformatic analysis in order to be transformed to understandable biological results. There are different forms of DNA-micro-arrays. Some are used for measuring gene expression, i.e. which genes are turned on and which genes are turned off. Others are used for sequencing, and finally some are used for measuring single nucleotide polymorphisms (SNPs). Some application possibilities of the arrays within the area of cell biology and classification of diseases are presented. Especially within the area of classification of cancer diseases the technology is promising, and a scientific breakthrough could occur in the area of classification of very aggressive cancers.

Computational Biology↗
PubMed2003

Technical evaluation of cDNA microarrays.

The aims were to evaluate the common reference design approach in microarray experiments and to evaluate the technical performance and the normalisation of cDNA microarrays with a limited number of spots. Total RNA from 3 normal and 3 tumour sample biopsies were used for synthesis of amino-allyl labelled cRNA. Equal amounts of cRNA from all samples were mixed as reference. After conjugation of cRNA with fluorophores (Cy3/Cy5), each individual tumour cRNA was hybridised to a cDNA microarray together with reference cRNA, scanned and analysed. We show that our procedures for producing cDNA microarrays are reproducible. The concordance between duplicated spots and replicate hybridisation was found to be high. We have demonstrated that our cDNA microarrays are of a high technical quality. The majority of the cDNA microarrays had low local spot background levels. Despite the high background levels for some local spots, variation could be minimized by locally weighted scatter plot smooth normalisation (LOWESS), which we showed was also suitable for normalisation of cDNA microarrays with a limited number of probes.

DNA, Complementary↗
PubMed2002

Antibody-based screening for hereditary nonpolyposis colorectal carcinoma compared with microsatellite analysis and sequencing.

BACKGROUND: Germline mutations in the DNA mismatch repair genes, MSH2, MLH1, and others are associated with hereditary nonpolyposis colorectal cancer (HNPCC). Due to the high costs of sequencing, cheaper screening methods are needed to identify HNPCC cases. Ideally, these methods should have a high sensitivity and identify all mutated cases without too many false-positive cases. METHODS: Sequencing was compared with microsatellite analysis and immunohistochemistry to detect the presence or absence of the mismatch repair proteins. In the current study, the authors examined 42 patients with colorectal carcinoma of whom 11 met the Amsterdam criteria and 31 were suspected to belong to HNPCC families. Thirty-five patients were examined by microsatellite analysis, 40 by immunohistochemical staining, and in 31 patients both the MLH1 and MSH2 genes were sequenced. RESULTS: Ninety-two percent of patients with germ line mutations were detected by either immunohistochemistry or microsatellite instability, indicating that a combination of these methods may be suitable for HNPCC screening. Microsatellite instability and abnormal immunohistochemical staining were found in 73% of the tumors. Concordance among the three methods was found in 74 % of the tumors. CONCLUSIONS: The authors suggest that immunohistochemistry should be used in combination with microsatellite analysis to prescreen suspected HNPCC patients for the selection of cases where sequencing of the MLH1 and MSH2 mismatch repair genes is indicated.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Generation of polyclonal plasmablasts from peripheral blood B cells: a normal counterpart of malignant plasmablasts.

A new way to identify tumor-specific genes is to compare gene expression profiles between malignant cells and their autologous normal counterparts. In patients with multiple myeloma, a major plasma cell disorder, normal plasma cells are not easily attainable in vivo. We report here that in vitro differentiation of peripheral blood B lymphocytes, purified from healthy donors and from patients with multiple myeloma, makes it possible to obtain a homogeneous population of normal plasmablastic cells. These cells were identified by their morphology, phenotype, production of polyclonal immunoglobulins, and expression of major transcription factors involved in B-cell differentiation. Oligonucleotide microarray analysis shows that these polyclonal plasmablastic cells have a gene expression pattern close to that of normal bone marrow-derived plasma cells. Detailed analysis of genes statistically differentially expressed between normal and tumor plasma cells allows the identification of myeloma-specific genes, including oncogenes and genes coding for tumor antigens. These data should help to disclose the molecular mechanisms of myeloma pathogenesis and to define new therapeutic targets in this still fatal malignancy. In addition, the comparison of gene expression between plasmablastic cells and B cells provides a new and powerful tool to identify genes specifically involved in normal plasma cell differentiation.

ADP-ribosyl Cyclase↗
PubMed2002

Immunohistochemical study of the expression of cell cycle regulating proteins at different stages of bladder cancer.

PURPOSE: The cell cycle is known to be deregulated in cancer. We therefore analyzed the expression of the cell cycle related proteins p21, p27, p16, Rb, and L-myc by immunohistochemical staining of bladder tumors. METHODS: The tissue material consisted of bladder tumors from three groups of patients; group 1, 23 patients with recurrent stage Ta (non-invasive) tumors; group 2, 22 patients presenting at their first admission with T2-4 (muscle invasive) tumors; group 3, 24 patients who experienced disease progression from Ta or T1 (invasive in connective tissue) to a higher stage. By immunohistochemical staining the protein expression was compared to allelic deletions of the corresponding genes. The allelic deletions were detected by PCR-based microsatellite analyses. RESULTS: We detected a significant reduction in the expression levels of the cell cycle related proteins p21(waf1) ( P=0.002), p27(kip1) ( P=0.03), Rb ( P=0.00002), and L-myc ( P=0.00000007) in muscle invasive tumors compared to noninvasive tumors. Tumors presenting as muscle invasive at first diagnosis had significantly lower levels of p16/CDKN2A ( P=0.01) when compared to muscle invasive tumors that followed Ta or T1 precursor lesions. We found no general correlation between allelic deletion of a gene and its immunohistochemical protein expression, indicating that the remaining allele may be capable of encoding a normal or even increased protein level. CONCLUSIONS: Our results support the hypothesis that bladder tumors with invasion at first diagnosis differ from those in which invasion follow superficial tumors. This difference is reflected as a different level in cell cycle related protein expression. The data also indicate that allelic deletions of cell cycle related genes do not correlate with an altered level of protein expression.

Cell Cycle Proteins↗
PubMed2002

Genome-wide study of gene copy numbers, transcripts, and protein levels in pairs of non-invasive and invasive human transitional cell carcinomas.

Gain and loss of chromosomal material is characteristic of bladder cancer, as well as malignant transformation in general. The consequences of these changes at both the transcription and translation levels is at present unknown partly because of technical limitations. Here we have attempted to address this question in pairs of non-invasive and invasive human bladder tumors using a combination of technology that included comparative genomic hybridization, high density oligonucleotide array-based monitoring of transcript levels (5600 genes), and high resolution two-dimensional gel electrophoresis. The results showed that there is a gene dosage effect that in some cases superimposes on other regulatory mechanisms. This effect depended (p < 0.015) on the magnitude of the comparative genomic hybridization change. In general (18 of 23 cases), chromosomal areas with more than 2-fold gain of DNA showed a corresponding increase in mRNA transcripts. Areas with loss of DNA, on the other hand, showed either reduced or unaltered transcript levels. Because most proteins resolved by two-dimensional gels are unknown it was only possible to compare mRNA and protein alterations in relatively few cases of well focused abundant proteins. With few exceptions we found a good correlation (p < 0.005) between transcript alterations and protein levels. The implications, as well as limitations, of the approach are discussed.

Carcinoma, Transitional Cell↗
PubMed2002

Proteomic strategies to reveal tumor heterogeneity among urothelial papillomas.

Proteomics and immunohistochemistry were used to reveal tumor heterogeneity among urothelial papillomas (UPs) with the long term goal of predicting their biological potential in terms of outcome. First, we identified proteins that were deregulated in invasive fresh lesions as compared with normal urothelium, and thereafter we immunostained UPs with a panel of antibodies against some of the markers. Twenty-two major proteins showing variations of 2-fold or more in at least one-third of the invasive lesions were selected. Specific antibodies against several of the proteins were obtained, but only a few reacted positively in immunostaining. A panel consisting of antibodies against keratinocytes (CKs) 5, 13, 18, and 20 and markers of squamous metaplasia (CKs 7, 8, and 14) was used to probe normal urothelium and 30 UPs collected during a period of five years. Four UPs showed a normal phenotype, whereas the rest could be grouped in five major types that shared aberrant staining with the CK20 antibody. Type 1 heterogeneity (n = 4) showed preferred staining of the umbrella cells with the CK8 antibody. Type 2 (n = 11) was typified by the staining of the basal and intermediate layers with the CK20 antibody. Type 3 (n = 7) was characterized by the predominant staining of the basal cell layer with the CK5 antibody. Type 4 (n = 1) showed areas of CK7 negative cells, whereas type 5 (n = 3) showed loss of staining of the basal cells with the CK20. 29% of the patients experienced recurrences, but none progressed to invasive disease. Patients harboring phenotypic alterations in the basal cell compartment (types 3 and 5) showed the highest number of recurrences (4/7 and 2/3, respectively), and all type 3 lesions progressed to a higher degree of dedifferentiation. Even though a long term prospective study involving a larger sample size is required to assess the biological potential of these lesions, we believe that this approach will prove instrumental for revealing early phenotypic changes in different types of cancer.

Antibodies↗