Abstract 1364
Cytosine methylation (mC) is a major DNA modification in higher eukaryotic genomes, which is involved in transcriptional silencing. A large amount of data has shown that patterns of DNA ...methylation are perturbed in hematological cancers including diffuse large B-cell lymphoma (DLBCL). The discovery that the TET hydroxylases convert mC to hydroxymethylcytosine (hmC) is a major break through for our understanding of how DNA methylation is deregulated. Multiple reports describe TET2 (Ten-Eleven Translocation 2) loss-of-function mutations in myeloid malignancies, and a recent study shows that TET2 inactivation perturbs both myeloid and lymphoid development in the mouse, and identifies TET2 mutations in ∼2% of human B-cell lymphoma (Quivoron et al, Cancer Cell 20, 1–14, 2011).
In the present study our aims are to determine the frequency and clinical impact of TET2 mutations in DLBCL, to identify TET2 target genes in CD34+ cells, normal- and malignant B-cells, and evaluate the role of TET2 mutations on the methylation pattern at TET2 targets genes in normal and malignant hematopoiesis.
DNA was isolated from fresh frozen DLBCL (n=110), normal CD34+ cells and B-cells, and a TET2 mutant DLBCL-cell line. Mutation scanning was performed by denaturing gradient gel electrophoresis (DGGE) and automated sequencing. Global methylation profiling was done by Illumina Infinium microarrays, methylation at individual genes by methylation specific melting curve analysis and pyrosequencing. Global mC and hmC patterns were determined by DNA immunoprecipitation and promoter array analysis in cell lines, B-cells and CD34+ cells. TET2 target genes were identified by ChIP followed by deep sequencing. Gene expression by Nimblegen custom made arrays and RT-qPCR.
We identified TET2 mutations in 15% of primary diffuse DLBCL, including missense mutation in the catalytic domain (n=8, 2 of which showed allelic loss), loss-of-function mutations (n=7, one of which showed allelic loss), and missense mutation outside the catalytic domain (n=1 with allelic loss). Somatic origin of these mutations was verified in 11 of the 16 cases where matched normal tissue was available. No difference in overall survival was observed between TET2mut and TET2wt cases (P=0.17). To a large extent, the TET2 targets genes identified by ChIP seq analysis were overlapping in CD34+ cells, normal- and malignant B-cells. Gene ontology analysis showed that TET2 target genes are mainly involved in DNA metabolism and repair, metabolic processes and cell cycle homeostasis. Global methylation in TET2mut and TET2wt cases and gene expression data are being analyzed in DLBCL samples. In addition, the distribution patterns of hmC and mC at TET2 target genes and the relation to gene expression is being analyzed in a TET2 mutant DLBCL cell line, normal B-cells and CD34+ cells.
Here, we show that TET2 mutations are frequent in DLBCL, and identify the TET2 target genes in CD34+ cells, and in normal and malignant B-cells. The role of TET2 mutations for global methylation and for the methylation patterns at TET2 target genes will be presented at the meeting. By investigating the clinical implications of TET2 mutations we aim to identify DLBCL subsets that may benefit from hypomethylating therapy. Furthermore, the identification of hypermethylated TET2 target genes will hopefully contribute to molecular understanding of how TET2 mutations induces malignant transformation.
Christensen:EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company. Helin:EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
With the realization that cancer is a genetic disease, detection of mutations in genomic DNA has become an important discipline in many areas of cancer research. Although the publication of the human ...genome sequence and the immense technological advancements have facilitated the analysis of cancer genomes, detection of mutations in tumor specimens may still be challenging and fraught with technical problems. In this review, we describe current technologies for detection of small DNA mutations, including mutation scanning techniques to search for unknown mutations, and diagnostic techniques to detect known cancer mutations. We outline the principles of the different techniques and discuss their advantages and limitations. We also discuss critical issues that must be considered before choosing methodology, including sensitivity, specificity, limit of detection, throughput and cost, quantity and quality of template DNA, available equipment, and personnel expertise.
50Detection of bladder cancer by analysis of urine DNA Serizawa, Reza R.; Ralfkiaer, Ulrik; Lam, Gitte W. ...
APMIS : acta pathologica, microbiologica et immunologica Scandinavica,
05/2008, Volume:
116, Issue:
5
Journal Article
Peer reviewed
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
34.
Detection of bladder cancer by analysis of urine DNA Serizawa, Reza R; Ralfkiaer, Ulrik; Lam, Gitte W ...
APMIS : acta pathologica, microbiologica et immunologica Scandinavica,
05/2008, Volume:
116, Issue:
5
Journal Article
Peer reviewed
Bladder cancer is a common cancer with 1500 new cases in Denmark every year. The most common bladder neoplasm is the transitional cell tumour, which presents either as a non-invasive or invasive form ...(transitiocellular carcinoma). Non-invasive tumours constitute around 50% of all newly diagnosed bladder tumours and although they are not as such life threatening they have a high recurrence rate of up to 80% and a progression rate up to 20%. Life long control is therefore mandatory. Control is done by cystoscopy which is uncomfortable for the patients, time and resource consuming. It is therefore optimal to develop a non-invasive test for detection of bladder cancer. In order to do this, we have systematically screened DNA from urine sediment and tumour biopsies from 120 primary bladder tumour patients and 40 control patients for mutations in FGFGR3, HRAS, KRAS, NRAS, PIK3CA and TP53. On the same patient material, we have also quantitatively determined the methylation status of a panel of 10 tumour suppressor genes. Our data shows that analysis of the mutational status of FGFR3 and PIK3CA combined with analysis of the methylation status of a representative panel of tumour suppressor genes might prove to be a useful non-invasive diagnostic test for bladder cancer.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Screening for copy number alterations (CNA) has improved by applying genome wide microarrays, where SNP-arrays also allow analysis of loss of heterozygosity (LOH). Currently, comparisons of high ...resolution microarray platforms are few, thus we performed a study to evaluate the power of differently designed microarrays for copy number analysis and LOH. We here analyzed 10 diagnostic chronic lymphocytic leukemia (CLL) samples (five IGVH mutated and five IGVH unmutated) using four different high-resolution platforms: BAC-arrays (32K), oligonucleotide-arrays (185K, Agilent), and two SNP-arrays (250K, Affymetrix and 317K, Illumina). Comparison of copy number data showed that the platforms are concordant in terms of detecting large CNA, including the known recurrent alterations. Mono-allelic and bi-allelic loss of 13q14 (3 and 1 sample, respectively), mono-allelic loss of 11q (1 sample), trisomy 12 (2 samples) and mono-allelic loss of 17p (2 samples) were concordant in all platforms. These aberrations were validated with FISH, which in addition identified subclones with mono-allelic loss of 13q14 in two cases, only detected with the BAC platform, rendering a cut-off for the power of detecting subclones to approximately 25% of investigated cells. As expected, all poor prognostic aberrations were detected in patients carrying unmutated IGHV genes whereas four of five mutated samples were detected with mono-allelic loss of 13q14, as the only recurrent alteration. Furthermore, detection of small CNA were in many cases discordant between platforms. Therefore, we defined alterations identified by at least two platforms and identified 47 losses and 31 gains using this criterion. We are currently validating the presence of a number of these alterations using other techniques. Evaluation of LOH showed concordance for 86 regions between the Illumina and Affymetrix platforms. Of these regions 12 LOH coincided with CNA, leaving the remaining 74 as copy-neutral LOH. In conclusion, all platforms investigated are powerful tools for screening of CNA, however, since non-overlapping CNA were detected by individual platforms, we emphasize the importance of validating findings. Also, there is a cut-off for detecting subclones, here estimated to 25%. Genomic arrays will improve the detection of new recurrent aberrations, which may potentially refine the prognostic hierarchy established by FISH.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Discovery-driven translational research in breast cancer is moving steadily from the study of cell lines to the analysis of clinically relevant samples that, together with the ever increasing number ...of novel and powerful technologies available within genomics, proteomics and functional genomics, promise to have a major impact on the way breast cancer will be diagnosed, treated and monitored in the future. Here we present a brief report on long-term ongoing strategies at the Danish Centre for Translational Breast Cancer Research to search for markers for early detection and targets for therapeutic intervention, to identify signalling pathways affected in individual tumours, as well as to integrate multiplatform 'omic' data sets collected from tissue samples obtained from individual patients. The ultimate goal of this initiative is to coalesce knowledge-based complementary procedures into a systems biology approach to fight breast cancer.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) ...technique allows simultaneous screening of multiple target sequences in a single reaction by using pairs of probes that carry tails for binding of common amplification primers. Resolution of the various targets is achieved by electrophoresis on the basis of predefined differences in amplicon length. In the conventional MLPA approach, one of the two target probes is generated by cloning in a single-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes will form a single amplifiable template whose length is defined by the number and lengths of the individual probes. We have used this principle to establish a methylation-specific MLPA (MS-MLPA) assay that simultaneously determines the methylation status of five promoter CpG islands, and we have used this assay to analyze DNA from tumor tissue and corresponding urine samples from patients with bladder cancer. Our data show that the use of multiple short synthetic probes provides a simple means for custom-designed MS-MLPA analysis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
50 Detection of bladder cancer by analysis of urine DNA Serizawa, Reza R.; Ralfkiær, Ulrik; Lam, Gitte W. ...
APMIS : acta pathologica, microbiologica et immunologica Scandinavica,
05/2008, Volume:
116, Issue:
5
Journal Article
Peer reviewed
Full text
Available for:
BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
To characterise clinicopathological features of mantle cell lymphoma (MCL) in the orbital and adnexal region.
Data on lymphoid lesions were retrieved searching the Danish Ocular Lymphoma Database ...1980-2005. Specimens were collected from Danish pathological departments and re-evaluated with a panel of monoclonal antibodies. For all patients with confirmed MCL the complete clinical files were collected and reviewed.
Twenty-one patients with MCL in the orbital and adnexal region were identified comprising 9% (21/230) of all lymphoma in the ocular region. There were 18 male patients and three female patients with an age range from 60 to 90 years (median 75 years). Orbital and adnexal region MCL as first presenting symptom comprised 67% of the patients. Of these, 71% had bilateral involvement. The orbit (71%) and eyelids (64%) were the most commonly affected sites. All but two presented in stage III/IV. Secondary MCL comprised 33% of the patients. Bilateral affection (29%) was less common in this patient group. The median survival was not different between the two presentation groups. Patients receiving anti-CD20 (rituximab)-containing chemotherapy had a significantly better 5-year overall survival (OS) rate (83%) than patients in treatment regimes without rituximab (5-year OS rate, 8%).
Orbital and adnexal region MCL presents in elderly males. The orbit and eyelid are frequently involved. There is a very high proportion of systemic involvement in general with MCL of the orbital and adnexal region. Most patients presented with stage IV disease and had multiple relapses and short survival time. Treatment with rituximab-containing chemotherapy improved survival significantly compared with combination chemotherapy without rituximab.
OBJECTIVE To characterize the clinicopathologic features of lacrimal gland lymphoma. METHODS All cases of lacrimal gland lymphoma from January 1, 1975, through December 31, 2009, were retrieved from ...The Danish Registry of Pathology. Histologic specimens were reevaluated using a panel of monoclonal antibodies. Clinical files from all patients with confirmed lymphoma were collected. RESULTS Twenty-seven patients with lacrimal gland lymphoma were identified. Eight of the patients were men and 19 were women; the median (range) age was 69 (43-87) years. The distribution of lymphoma subtypes was as follows: extranodal marginal zone lymphoma, 10 (37%); follicular lymphoma, 5 (19%); diffuse large B-cell lymphoma, 4 (15%); mantle cell lymphoma, 3 (11%); chronic lymphocytic leukemia/small lymphatic lymphoma, 2 (7%); and unclassified B-cell lymphoma, 3 (11%). Twenty-two patients (81%) had stage I or II lymphoma, 1 patient (4%) had stage III lymphoma, and 4 patients (15%) had stage IV lymphoma. Patients with stage I or II lymphoma were treated with radiotherapy (15 67%), chemotherapy (3 14%), chemotherapy plus radiotherapy (1 5%), and surgery (3 14%). Patients presenting with stage III or IV lymphoma were treated with chemotherapy alone. Complete remission was observed in 23 of the patients (85%), although 12 (44%) of these had a relapse, independent of subtype, stage, or treatment. The 5-year overall survival was 70%. CONCLUSIONS Malignant lymphoma of the lacrimal gland is relatively rare and is mostly prevalent in elderly women. The distribution of lacrimal gland lymphoma subtypes resembles that of lymphoma subtypes of the salivary glands. The majority of lacrimal gland lymphomas are low grade, and the prognosis is relatively good.
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