Ulrik Ralfkiaer

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL)

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.

Integrated genetic and epigenetic analysis of bladder cancer reveals an additive diagnostic value of FGFR3 mutations and hypermethylation events.

The bladder cancer genome harbors numerous oncogenic mutations and aberrantly methylated gene promoters. The aim of our study was to generate a profile of these alterations and investigate their use as biomarkers in urine sediments for noninvasive detection of bladder cancer. We systematically screened FGFR3, PIK3CA, TP53, HRAS, NRAS and KRAS for mutations and quantitatively assessed the methylation status of APC, ARF, DBC1, INK4A, RARB, RASSF1A, SFRP1, SFRP2, SFRP4, SFRP5 and WIF1 in a prospective series of tumor biopsies (N = 105) and urine samples (N = 113) from 118 bladder tumor patients. We also analyzed urine samples from 33 patients with noncancerous urinary lesions. A total of 95 oncogenic mutations and 189 hypermethylation events were detected in the 105 tumor biopsies. The total panel of markers provided a sensitivity of 93%, whereas mutation and methylation markers alone provided sensitivities of 72% and 70%, respectively. In urine samples, the sensitivity was 70% for all markers, 50% for mutation markers and 52% for methylation markers. FGFR3 mutations occurred more frequently in tumors with no methylation events than in tumors with one or more methylation events (78% vs. 33%; p < 0.0001). FGFR3 mutation in combination with three methylation markers (APC, RASSF1A and SFRP2) provided a sensitivity of 90% in tumors and 62% in urine with 100% specificity. These results suggest an inverse correlation between FGFR3 mutations and hypermethylation events, which may be used to improve noninvasive, DNA‐based detection of bladder cancer.

Malignant cutaneous T-cell lymphoma cells express IL-17 utilizing the Jak3/Stat3 signaling pathway.

IL-17 is a proinflammatory cytokine that is crucial for the hosts protection against a range of extracellular pathogens. However, inappropriately regulated expression of IL-17 is associated with the development of inflammatory diseases and cancer. In cutaneous T-cell lymphoma (CTCL), malignant T cells gradually accumulate in skin lesions characterized by massive chronic inflammation, suggesting that IL-17 could be involved in the pathogenesis. In this study we show that IL-17 protein is present in 10 of 13 examined skin lesions but not in sera from 28 CTCL patients. Importantly, IL-17 expression is primarily observed in atypical lymphocytes with characteristic neoplastic cell morphology. In accordance, malignant T-cell lines from CTCL patients produce IL-17 and the synthesis is selectively increased by IL-2 receptor β chain cytokines. Small-molecule inhibitors or small interfering RNA against Jak3 and signal transducer and activator of transcription 3 (Stat3) reduce the production of IL-17, showing that the Jak3/Stat3 pathway promotes the expression of the cytokine. In summary, our findings indicate that the malignant T cells in CTCL lesions express IL-17 and that this expression is promoted by the Jak3/Stat3 pathway.

STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains elusive. Recent discoveries indicate that the oncogenic microRNA miR-155 is overexpressed in affected skin from CTCL patients. Here, we address what drives the expression of miR-155 and investigate its role in the pathogenesis of CTCL. We show that malignant T cells constitutively express high levels of miR-155 and its host gene BIC (B cell integration cluster). Using ChIP-seq, we identify BIC as a target of transcription factor STAT5, which is aberrantly activated in malignant T cells and induced by IL-2/IL-15 in non-malignant T cells. Incubation with JAK inhibitor or siRNA-mediated knockdown of STAT5 decreases BIC/miR-155 expression, whereas IL-2 and IL-15 increase their expression in cell lines and primary cells. In contrast, knockdown of STAT3 has no effect, and BIC is not a transcriptional target of STAT3, indicating that regulation of BIC/miR-155 expression by STAT5 is highly specific. Malignant proliferation is significantly inhibited by an antisense-miR-155 as well as by knockdown of STAT5 and BIC. In conclusion, we provide the first evidence that STAT5 drives expression of oncogenic BIC/miR-155 in cancer. Moreover, our data indicate that the STAT5/BIC/miR-155 pathway promotes proliferation of malignant T cells, and therefore is a putative target for therapy in CTCL.

Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma

The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P<0.0001), and at CpG-rich promoters (60%; P<0.0001) of genes involved in hematopoietic differentiation and cellular development. Hypermethylated loci in TET2 mutated samples overlap with the bivalent (H3K27me3/H3K4me3) silencing mark in human embryonic stem cells (P=1.5×10−30). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells.

Towards discovery‐driven translational research in breast cancer

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.

Equitoxic doses of 5-azacytidine and 5-aza-2'deoxycytidine induce diverse immediate and overlapping heritable changes in the transcriptome.

Background The hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2′deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT). While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA), which may potentially affect the activities of the drugs in patients. Methodology/Principal Findings A hematopoietic (HL-60; acute myeloid leukemia) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2) and lasting (day 8) effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells. Conclusions/Significance Overall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.

Frequent hypermethylation of DBC1 in malignant lymphoproliferative neoplasms.

Allelic loss at chromosome 9q31–34 is a frequent event in many lymphoproliferative malignancies. Here, we examined DBC1 at 9q33.1 as a potential target in lymphomagenesis. DBC1 is a putative tumor suppressor that has been shown to be involved in the regulation of cell growth and programmed cell death. The methylation status of the DBC1 promoter CpG island was examined by methylation-specific PCR, bisulfite sequencing, and methylation-specific melting curve analysis. DBC1 was hypermethylated in 5 of 5 B-cell-derived lymphoma cell lines, 41 of 42 diffuse large B-cell lymphomas, 24 of 24 follicular lymphomas, 5 of 5 mantle cell lymphomas, 4 of 4 small lymphocytic lymphomas, 1 of 2 lymphoplasmacytoid lymphomas, and in 12 of 12 acute lymphoblastic leukemias, but was unmethylated in 1 case of splenic marginal zone lymphoma, in 12 of 12 multiple myelomas, in 24 of 24 reactive lymph nodes, and in 12 of 12 samples of blood lymphocytes from random donors. DBC1 hypermethylation was associated with transcriptional silencing in lymphoma cell lines, and reexpression of this gene could be induced by treatment with the demethylating agent, 5-aza-2′-deoxycytidine. Our data suggest that hypermethylation of the DBC1 promoter region is a frequent event during the development of lymphoproliferative malignancies, and that DBC1 hypermethylation may serve as a marker for these cancers.

Expression of miR-155 and miR-126 in situ in cutaneous T-cell lymphoma.

Recently, miR‐155 has been implicated in cutaneous T‐cell lymphoma (CTCL). Thus, elevated levels of miR‐155 were observed in skin lesions from CTCL patients as judged from qPCR and micro‐array analysis and aberrant, high miR‐155 expression was associated with severe disease. Moreover, miR‐155 promoted proliferation of malignant T cells in vitro. Little is, however, known about which cell types express miR‐155 in vivo in CTCL skin lesions. Here, we study miR‐155 expression using in situ hybridization (ISH) with a miR‐155 probe, a negative control (scrambled), and a miR‐126 probe as a positive control in nine patients with mycosis fungoides, the most frequent subtype of CTCL. We provide evidence that both malignant and non‐malignant T cells stain weakly to moderately positive with the miR‐155 probe, but generally negative with the miR‐126 and negative control probes. Reversely, endothelial cells stain positive for miR‐126 and negative for miR‐155 and the control probe. Solitary T cells with a malignant morphology display brighter staining with the miR‐155 probe. Taken together, our findings suggest that both malignant and non‐malignant T cells express miR‐155 in situ in CTCL. Moreover, they indicate heterogeneity in miR‐155 expression among malignant T cells.

Custom-Designed MLPA Using Multiple Short Synthetic Probes: Application to Methylation Analysis of Five Promoter CpG Islands in Tumor and Urine Specimens from Patients with Bladder Cancer

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.