Simon Husby, Francesco Favero, Francisco G. Rodriguez-Gonzalez, Lesley A. Sutton, Eva K. Haastrup... more Simon Husby, Francesco Favero, Francisco G. Rodriguez-Gonzalez, Lesley A. Sutton, Eva K. Haastrup, Andreas Due Ørskov, Jakob W. Hansen, Bente Arboe, Derya Aslan, Erik Clasen-Linde, Lise Mette Rahbek Gjerdrum, Jette Sønderskov Gørlev, Peter Brown, Anne Fischer-Nielsen, Richard Rosenquist, Joachim Weischenfeldt and Kirsten Grønbæk Department of Hematology, Rigshospitalet, Copenhagen, Denmark; Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark; Faculty of Health Sciences, Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, University of Copenhagen, Copenhagen, Denmark; Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark; Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; Department of Clinical Immunology, Rigshospitalet, Copenhagen, Denmark; Department of Pathology, Rigshospitalet, Copenhagen, Denmark; Department of Pathology, Zealand University Hospital, Roskilde, Denmark
Staphylococcus aureus enterotoxins (SE) are believed to fuel disease activity in cutaneous T-cell... more Staphylococcus aureus enterotoxins (SE) are believed to fuel disease activity in cutaneous T-cell lymphoma (CTCL). Recent data support this by showing that antibiotics inhibit malignant T cells in skin lesions in mycosis fungoides and Sezary syndrome, the most common forms of CTCL. Yet, it remains incompletely characterized how SE fuel disease activity. Here, we show that SE induce expression of the oncogenic microRNA mir-155 in primary malignant T cells. Thus, SE and S. aureus-isolates from lesional patient skin induce mir-155 expression, at least partly, through the IL-2Rg/JAK/STAT5 pathway, and the effect is augmented by the presence of non-malignant T cells. Importantly, mycosis fungoides lesions harbor S. aureus, express pY-STAT5, and display enhanced mir-155 expression, when compared with non-lesional and healthy skin. Preliminary data show that aggressive antibiotic therapy is associated with decreased pY-STAT5 and mir-155 expression in lesional skin in two patients with Sezary syndrome. In conclusion, we demonstrate that S. aureus and its enterotoxins induce enhanced expression of oncogenic mir-155 providing mechanistic insight into the role of S. aureus in CTCL. Our findings support that environmental stimuli such as bacteria can fuel disease progression in CTCL.
PurposeThe Danish Pathology Life Course (PATHOLIFE) cohort was established to facilitate epidemio... more PurposeThe Danish Pathology Life Course (PATHOLIFE) cohort was established to facilitate epidemiological research relating histological and cytological features extracted from patient tissue specimens to the rich life course histories, including both prior and future register data, of the entire Danish population. Research results may increase quality of diagnosis, prognosis and stratification of patient subtypes, possibly identifying novel routes of treatment.ParticipantsAll Danish residents from 1 January 1986 to 31 December 2019, totalling 8 593 421 individuals.Findings to dateWe provide an overview of the subpopulation of Danish residents who have had a tissue specimen investigated within the Danish healthcare system, including both the primary sector and hospitals. We demonstrate heterogeneity in sociodemographic and prognostic factors between the general Danish population and the above mentioned subpopulation, and also between the general Danish population and subpopulations o...
ABSTRACTDefining the molecular phenotype of single cellsin-situis essential for understanding tis... more ABSTRACTDefining the molecular phenotype of single cellsin-situis essential for understanding tissue heterogeneity in health and disease. Powerful imaging technologies have recently been joined by spatial omics technologies, promising unparalleled insights into the molecular landscape of biological samples. One approach involves laser microdissection in combination with membrane glass slides for the isolation of single cells from specific anatomical regions for further analysis by spatial omics. However, so far this is not fully compatible with automated staining platforms and routine histology procedures such as heat-induced epitope retrieval, limiting reproducibility, throughput and integration of advanced staining procedures. This study describes a robust workflow for routine use of glass membrane slides, allowing precise extraction of tissue in combination with automated and multicolor immunofluorescence staining. The key advance is the addition of glycerol to standard heat-indu...
Our understanding of the regulatory processes of reepithelialization during wound healing is inco... more Our understanding of the regulatory processes of reepithelialization during wound healing is incomplete. In an attempt to map the genes involved in epidermal regeneration and differentiation, we measured gene expression in formalin-fixed, paraffin-embedded standardized epidermal wounds induced by the suction-blister technique with associated nonwounded skin using NanoString technology. The transcripts of 139 selected genes involved in clotting, immune response to tissue injury, signaling pathways, cell adhesion and proliferation, extracellular matrix remodeling, zinc transport and keratinocyte differentiation were evaluated. We identified 22 upregulated differentially expressed genes (DEGs) in descending order of fold change (MMP1, MMP3, IL6, CXCL8, SERPINE1, IL1B, PTGS2, HBEGF, CXCL5, CXCL2, TIMP1, CYR61, CXCL1, MMP12, MMP9, HGF, CTGF, ITGB3, MT2A, FGF7, COL4A1 and PLAUR). The expression of the most upregulated gene, MMP1, correlated strongly with MMP3 followed by IL6 and IL1B. rhI...
Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumu... more Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms that hamper quality of life and are entry sites for bacterial infection, a major cause of morbidity and mortality in advanced diseases. The mechanism driving the pathological processes that compromise the skin barrier remains unknown. Here, we report increased transepidermal water loss and compromised expression of the skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX-positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as interleukin 13 [IL-13], IL-22, and oncostatin M) that activate STAT3 signaling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, the repression of filaggrins can be counteracted by a cocktail of antibodies targeting these cytokines/...
scape and its impact in multiple myeloma. Nat Commun. 2017;8(1):1893. 3. Foltz SM, Gao Q, Yoon CJ... more scape and its impact in multiple myeloma. Nat Commun. 2017;8(1):1893. 3. Foltz SM, Gao Q, Yoon CJ, et al. Evolution and structure of clinically relevant gene fusions in multiple myeloma. Nat Commun. 2020;11(1):2666. 4. Carrasco-Leon A, Ezponda T, Meydan C, et al. Characterization of complete lncRNAs transcriptome reveals the functional and clinical impact of lncRNAs in multiple myeloma. Leukemia. 2021;35(5):1438-1450. 5. Agirre X, Meydan C, Jiang Y, et al. Long non-coding RNAs discriminate the stages and gene regulatory states of human humoral immune response. Nat Commun. 2019;10(1):821. 6. Chakravarthi BV, Dedigama-Arachchige P, Carskadon S, et al. Pseudogene associated recurrent gene fusion in prostate cancer. Neoplasia. 2019;21(10):989-1002.
Most patients cannot be included in randomized clinical trials. We report real-world outcomes of ... more Most patients cannot be included in randomized clinical trials. We report real-world outcomes of all Danish patients with multiple myeloma (MM) treated with daratumumab-based regimens until 1 January 2019. Methods Information of 635 patients treated with daratumumab was collected retrospectively and included lines of therapy (LOT), hematologic responses according to the International Myeloma Working Group recommendations, time to next treatment (TNT) and the cause of discontinuation of treatment. Baseline characteristics were acquired from the validated Danish Multiple Myeloma Registry (DMMR). Results Daratumumab was administrated as monotherapy (Da-mono) in 27.7%, in combination with immunomodulatory drugs (Da-IMiD) in 57.3%, in combination with proteasome inhibitors (Da-PI) in 11.2% and in other combinations (Da-other) in 3.8% of patients. The median number of lines of therapy given before daratumumab was 5 for Da-mono, 3 for Da-IMiD, 4 for Da-PI, and 2 for Da-other. In Da-mono, o...
BackgroundThe calreticulin (CALR) exon 9 mutations that are identified in 20% of patients with Ph... more BackgroundThe calreticulin (CALR) exon 9 mutations that are identified in 20% of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN) generate immunogenic antigens. Thus, therapeutic cancer vaccination against mutant CALR could be a new treatment modality in CALR-mutant MPN.MethodsThe safety and efficacy of vaccination with the peptide CALRLong36 derived from the CALR exon 9 mutations was tested in a phase I clinical vaccination trial with montanide as adjuvant. Ten patients with CALRmut MPN were included in the trial and received 15 vaccines over the course of one year. The primary end point was evaluation of safety and toxicity of the vaccine. Secondary endpoint was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT03566446).ResultsPatients had a median age of 59.5 years and a median disease duration of 6.5 years. All patients received the intended 15 vaccines, and the vaccines were deemed safe a...
ABSTRACTThe systems-wide analysis of biomolecules in time and space is key to our understanding o... more ABSTRACTThe systems-wide analysis of biomolecules in time and space is key to our understanding of cellular function and heterogeneity in health and disease1. Remarkable technological progress in microscopy and multi-omics technologies enable increasingly data-rich descriptions of tissue heterogeneity2,3,4,5. Single cell sequencing, in particular, now routinely allows the mapping of cell types and states uncovering tremendous complexity6. Yet, an unaddressed challenge is the development of a method that would directly connect the visual dimension with the molecular phenotype and in particular with the unbiased characterization of proteomes, a close proxy for cellular function. Here we introduce Deep Visual Proteomics (DVP), which combines advances in artificial intelligence (AI)-driven image analysis of cellular phenotypes with automated single cell laser microdissection and ultra-high sensitivity mass spectrometry7. DVP links protein abundance to complex cellular or subcellular phe...
We report the final 2-year end-of-study results from the first clinical trial investigating combi... more We report the final 2-year end-of-study results from the first clinical trial investigating combination treatment with ruxolitinib and low-dose pegylated interferon-α2 (PEG-IFNα2). The study included 32 patients with polycythemia vera and 18 with primary or secondary myelofibrosis; 46 patients were previously intolerant of or refractory to PEGIFNα2. The primary outcome was efficacy, based on hematologic parameters, quality of life measurements, and JAK2 V617F allele burden. We used the 2013 European LeukemiaNet and International Working Group- Myeloproliferative Neoplasms Research and Treatment response criteria, including response in symptoms, splenomegaly, peripheral blood counts, and bone marrow. Of 32 patients with polycythemia vera, ten (31%) achieved a remission which was a complete remission in three (9%) cases. Of 18 patients with myelofibrosis, eight (44%) achieved a remission; five (28%) were complete remissions. The cumulative incidence of peripheral blood count remission...
Simon Husby, Francesco Favero, Francisco G. Rodriguez-Gonzalez, Lesley A. Sutton, Eva K. Haastrup... more Simon Husby, Francesco Favero, Francisco G. Rodriguez-Gonzalez, Lesley A. Sutton, Eva K. Haastrup, Andreas Due Ørskov, Jakob W. Hansen, Bente Arboe, Derya Aslan, Erik Clasen-Linde, Lise Mette Rahbek Gjerdrum, Jette Sønderskov Gørlev, Peter Brown, Anne Fischer-Nielsen, Richard Rosenquist, Joachim Weischenfeldt and Kirsten Grønbæk Department of Hematology, Rigshospitalet, Copenhagen, Denmark; Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark; Faculty of Health Sciences, Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, University of Copenhagen, Copenhagen, Denmark; Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark; Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; Department of Clinical Immunology, Rigshospitalet, Copenhagen, Denmark; Department of Pathology, Rigshospitalet, Copenhagen, Denmark; Department of Pathology, Zealand University Hospital, Roskilde, Denmark
Staphylococcus aureus enterotoxins (SE) are believed to fuel disease activity in cutaneous T-cell... more Staphylococcus aureus enterotoxins (SE) are believed to fuel disease activity in cutaneous T-cell lymphoma (CTCL). Recent data support this by showing that antibiotics inhibit malignant T cells in skin lesions in mycosis fungoides and Sezary syndrome, the most common forms of CTCL. Yet, it remains incompletely characterized how SE fuel disease activity. Here, we show that SE induce expression of the oncogenic microRNA mir-155 in primary malignant T cells. Thus, SE and S. aureus-isolates from lesional patient skin induce mir-155 expression, at least partly, through the IL-2Rg/JAK/STAT5 pathway, and the effect is augmented by the presence of non-malignant T cells. Importantly, mycosis fungoides lesions harbor S. aureus, express pY-STAT5, and display enhanced mir-155 expression, when compared with non-lesional and healthy skin. Preliminary data show that aggressive antibiotic therapy is associated with decreased pY-STAT5 and mir-155 expression in lesional skin in two patients with Sezary syndrome. In conclusion, we demonstrate that S. aureus and its enterotoxins induce enhanced expression of oncogenic mir-155 providing mechanistic insight into the role of S. aureus in CTCL. Our findings support that environmental stimuli such as bacteria can fuel disease progression in CTCL.
PurposeThe Danish Pathology Life Course (PATHOLIFE) cohort was established to facilitate epidemio... more PurposeThe Danish Pathology Life Course (PATHOLIFE) cohort was established to facilitate epidemiological research relating histological and cytological features extracted from patient tissue specimens to the rich life course histories, including both prior and future register data, of the entire Danish population. Research results may increase quality of diagnosis, prognosis and stratification of patient subtypes, possibly identifying novel routes of treatment.ParticipantsAll Danish residents from 1 January 1986 to 31 December 2019, totalling 8 593 421 individuals.Findings to dateWe provide an overview of the subpopulation of Danish residents who have had a tissue specimen investigated within the Danish healthcare system, including both the primary sector and hospitals. We demonstrate heterogeneity in sociodemographic and prognostic factors between the general Danish population and the above mentioned subpopulation, and also between the general Danish population and subpopulations o...
ABSTRACTDefining the molecular phenotype of single cellsin-situis essential for understanding tis... more ABSTRACTDefining the molecular phenotype of single cellsin-situis essential for understanding tissue heterogeneity in health and disease. Powerful imaging technologies have recently been joined by spatial omics technologies, promising unparalleled insights into the molecular landscape of biological samples. One approach involves laser microdissection in combination with membrane glass slides for the isolation of single cells from specific anatomical regions for further analysis by spatial omics. However, so far this is not fully compatible with automated staining platforms and routine histology procedures such as heat-induced epitope retrieval, limiting reproducibility, throughput and integration of advanced staining procedures. This study describes a robust workflow for routine use of glass membrane slides, allowing precise extraction of tissue in combination with automated and multicolor immunofluorescence staining. The key advance is the addition of glycerol to standard heat-indu...
Our understanding of the regulatory processes of reepithelialization during wound healing is inco... more Our understanding of the regulatory processes of reepithelialization during wound healing is incomplete. In an attempt to map the genes involved in epidermal regeneration and differentiation, we measured gene expression in formalin-fixed, paraffin-embedded standardized epidermal wounds induced by the suction-blister technique with associated nonwounded skin using NanoString technology. The transcripts of 139 selected genes involved in clotting, immune response to tissue injury, signaling pathways, cell adhesion and proliferation, extracellular matrix remodeling, zinc transport and keratinocyte differentiation were evaluated. We identified 22 upregulated differentially expressed genes (DEGs) in descending order of fold change (MMP1, MMP3, IL6, CXCL8, SERPINE1, IL1B, PTGS2, HBEGF, CXCL5, CXCL2, TIMP1, CYR61, CXCL1, MMP12, MMP9, HGF, CTGF, ITGB3, MT2A, FGF7, COL4A1 and PLAUR). The expression of the most upregulated gene, MMP1, correlated strongly with MMP3 followed by IL6 and IL1B. rhI...
Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumu... more Cutaneous T-cell lymphoma (CTCL) is a devastating lymphoid malignancy characterized by the accumulation of malignant T cells in the dermis and epidermis. Skin lesions cause serious symptoms that hamper quality of life and are entry sites for bacterial infection, a major cause of morbidity and mortality in advanced diseases. The mechanism driving the pathological processes that compromise the skin barrier remains unknown. Here, we report increased transepidermal water loss and compromised expression of the skin barrier proteins filaggrin and filaggrin-2 in areas adjacent to TOX-positive T cells in CTCL skin lesions. Malignant T cells secrete mediators (including cytokines such as interleukin 13 [IL-13], IL-22, and oncostatin M) that activate STAT3 signaling and downregulate filaggrin and filaggrin-2 expression in human keratinocytes and reconstructed human epithelium. Consequently, the repression of filaggrins can be counteracted by a cocktail of antibodies targeting these cytokines/...
scape and its impact in multiple myeloma. Nat Commun. 2017;8(1):1893. 3. Foltz SM, Gao Q, Yoon CJ... more scape and its impact in multiple myeloma. Nat Commun. 2017;8(1):1893. 3. Foltz SM, Gao Q, Yoon CJ, et al. Evolution and structure of clinically relevant gene fusions in multiple myeloma. Nat Commun. 2020;11(1):2666. 4. Carrasco-Leon A, Ezponda T, Meydan C, et al. Characterization of complete lncRNAs transcriptome reveals the functional and clinical impact of lncRNAs in multiple myeloma. Leukemia. 2021;35(5):1438-1450. 5. Agirre X, Meydan C, Jiang Y, et al. Long non-coding RNAs discriminate the stages and gene regulatory states of human humoral immune response. Nat Commun. 2019;10(1):821. 6. Chakravarthi BV, Dedigama-Arachchige P, Carskadon S, et al. Pseudogene associated recurrent gene fusion in prostate cancer. Neoplasia. 2019;21(10):989-1002.
Most patients cannot be included in randomized clinical trials. We report real-world outcomes of ... more Most patients cannot be included in randomized clinical trials. We report real-world outcomes of all Danish patients with multiple myeloma (MM) treated with daratumumab-based regimens until 1 January 2019. Methods Information of 635 patients treated with daratumumab was collected retrospectively and included lines of therapy (LOT), hematologic responses according to the International Myeloma Working Group recommendations, time to next treatment (TNT) and the cause of discontinuation of treatment. Baseline characteristics were acquired from the validated Danish Multiple Myeloma Registry (DMMR). Results Daratumumab was administrated as monotherapy (Da-mono) in 27.7%, in combination with immunomodulatory drugs (Da-IMiD) in 57.3%, in combination with proteasome inhibitors (Da-PI) in 11.2% and in other combinations (Da-other) in 3.8% of patients. The median number of lines of therapy given before daratumumab was 5 for Da-mono, 3 for Da-IMiD, 4 for Da-PI, and 2 for Da-other. In Da-mono, o...
BackgroundThe calreticulin (CALR) exon 9 mutations that are identified in 20% of patients with Ph... more BackgroundThe calreticulin (CALR) exon 9 mutations that are identified in 20% of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN) generate immunogenic antigens. Thus, therapeutic cancer vaccination against mutant CALR could be a new treatment modality in CALR-mutant MPN.MethodsThe safety and efficacy of vaccination with the peptide CALRLong36 derived from the CALR exon 9 mutations was tested in a phase I clinical vaccination trial with montanide as adjuvant. Ten patients with CALRmut MPN were included in the trial and received 15 vaccines over the course of one year. The primary end point was evaluation of safety and toxicity of the vaccine. Secondary endpoint was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT03566446).ResultsPatients had a median age of 59.5 years and a median disease duration of 6.5 years. All patients received the intended 15 vaccines, and the vaccines were deemed safe a...
ABSTRACTThe systems-wide analysis of biomolecules in time and space is key to our understanding o... more ABSTRACTThe systems-wide analysis of biomolecules in time and space is key to our understanding of cellular function and heterogeneity in health and disease1. Remarkable technological progress in microscopy and multi-omics technologies enable increasingly data-rich descriptions of tissue heterogeneity2,3,4,5. Single cell sequencing, in particular, now routinely allows the mapping of cell types and states uncovering tremendous complexity6. Yet, an unaddressed challenge is the development of a method that would directly connect the visual dimension with the molecular phenotype and in particular with the unbiased characterization of proteomes, a close proxy for cellular function. Here we introduce Deep Visual Proteomics (DVP), which combines advances in artificial intelligence (AI)-driven image analysis of cellular phenotypes with automated single cell laser microdissection and ultra-high sensitivity mass spectrometry7. DVP links protein abundance to complex cellular or subcellular phe...
We report the final 2-year end-of-study results from the first clinical trial investigating combi... more We report the final 2-year end-of-study results from the first clinical trial investigating combination treatment with ruxolitinib and low-dose pegylated interferon-α2 (PEG-IFNα2). The study included 32 patients with polycythemia vera and 18 with primary or secondary myelofibrosis; 46 patients were previously intolerant of or refractory to PEGIFNα2. The primary outcome was efficacy, based on hematologic parameters, quality of life measurements, and JAK2 V617F allele burden. We used the 2013 European LeukemiaNet and International Working Group- Myeloproliferative Neoplasms Research and Treatment response criteria, including response in symptoms, splenomegaly, peripheral blood counts, and bone marrow. Of 32 patients with polycythemia vera, ten (31%) achieved a remission which was a complete remission in three (9%) cases. Of 18 patients with myelofibrosis, eight (44%) achieved a remission; five (28%) were complete remissions. The cumulative incidence of peripheral blood count remission...
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