Publications by authors named "Håkan Mellstedt"

104 Publications

Targeting the Receptor Tyrosine Kinase ROR1 by Small Molecules.

Handb Exp Pharmacol 2021 Sep 7. Epub 2021 Sep 7.

BioClinicum, Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.

Receptor tyrosine kinases (RTKs) are frequently dysregulated in malignancies and important for the malignant characteristics of tumor cells. RTKs are attractive structures for drug targeting of cancer. The RTK ROR1 is of significance during embryogenesis but downregulated in post-partum tissues. However, ROR1 is overexpressed in several hematological and solid tumors and important for tumor cell proliferation, survival, migration, and metastasis. WNT5a is a main ligand for ROR1. Several clinical trials are ongoing using anti-ROR1 antibody based drugs directed against the external domain (monoclonal antibodies, BiTE, CAR-T). We have produced small molecules (KAN834/1571c) fitting to the ATP pocket of the intracellular tyrosine kinase (TK) domain of ROR1 (TK inhibitor, TKI). These inhibitors of ROR1 prevented ROR1 phosphorylation and inactivated the WNT/β-catenin independent as well as WNT/β-catenin dependent pathways. ROR1-TKI induced apoptosis of ROR1 positive fresh patient derived tumor cells and appropriate cell lines and a dose and time dependent tumor reduction in animal models. In combination with other clinically relevant targeting drugs as venetoclax a synergistic apoptotic effect was seen. Two other small molecules (ARI-1 and strictinin) bound also to ROR1 and inhibited tumor growth. Development of small molecule ROR1 inhibitors is warranted to include this novel therapeutic approach for cancer therapy.
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http://dx.doi.org/10.1007/164_2021_535DOI Listing
September 2021

Predicting ROR1/BCL2 combination targeted therapy of small cell carcinoma of the lung.

Cell Death Dis 2021 06 4;12(6):577. Epub 2021 Jun 4.

Department of Internal Medicine, Division of Medical Oncology, The Ohio State University, Columbus, OH, 43210, USA.

Small cell lung cancer (SCLC) remains a deadly form of cancer, with a 5-year survival rate of less than 10 percent, necessitating novel therapies. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is emerging as a therapeutic target and is co-expressed with BCL2 in multiple tumor types due to microRNA coregulation. We hypothesize that ROR1-targeted therapy is effective in small cell lung cancer and synergizes with therapeutic BCL2 inhibition. Tissue microarrays (TMAs) and formalin-fixed paraffin-embedded (FFPE) SCLC patient samples were utilized to determine the prevalence of ROR1 and BCL2 expression in SCLC. Eight SCLC-derived cell lines were used to determine the antitumor activity of a small molecule ROR1 inhibitor (KAN0441571C) alone and in combination with the BCL2 inhibitor venetoclax. The Chou-Talalay method was utilized to determine synergy with the drug combination. ROR1 and BCL2 protein expression was identified in 93% (52/56) and 86% (48/56) of SCLC patient samples, respectively. Similarly, ROR1 and BCL2 were shown by qRT-PCR to have elevated expression in 79% (22/28) and 100% (28/28) of SCLC patient samples, respectively. KAN0441571C displayed efficacy in 8 SCLC cell lines, with an IC50 of 500 nM or less. Synergy as defined by a combination index of <1 via the Chou-Talalay method between KAN0441571C and venetoclax was demonstrated in 8 SCLC cell lines. We have shown that ROR1 inhibition is synergistic with BCL2 inhibition in SCLC models and shows promise as a novel therapeutic target in SCLC.
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http://dx.doi.org/10.1038/s41419-021-03855-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178315PMC
June 2021

Polymorphisms within methotrexate pathway genes: Relationship between plasma methotrexate levels, toxicity experienced and outcome in pediatric acute lymphoblastic leukemia.

Iran J Basic Med Sci 2020 Jun;23(6):800-809

Department of Hematology, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran.

Objectives: The current study aimed to investigate the relationship of genetic polymorphism and plasma methotrexate (MTX) levels, toxicity experience and event free survival (EFS) in pediatric acute lymphoblastic leukemia (ALL).

Materials And Methods: The study included 74 ALL patients. Polymerase chain reaction and genotyping of methylene tetrahydrofolate reductase (MTHFR) rs1801133, MTHFR rs1801131, ATP-binding cassette superfamily B1 (ABCB1) rs1045642, ATP-binding cassette superfamily G2 (ABCG2) rs2231142 and solute carrier 19A1 (SLC19A1) rs1051266 genetic variations were performed. The plasma MTX levels were investigated at 48 hr after the first dose of MTX infusion.

Results: MTHFR rs1801133 TT genotype, ABCBa1 rs1045642 CT genotype and ABCG2 rs2231142 CA genotype revealed a statistically significant association with the MTX plasma levels (<0.01, <0.05, <0.05, respectively). The MTHFR rs1801133 TT genotype had a statistically significant association with hematopoietic toxicity (<0.01) and interventions (<0.05). The MTHFR rs1801131 AC genotype was related to the decreased hepatic toxicity (<0.05). The SLC19A1 rs 1051266 GA genotype was related to the increased hepatic toxicity (<0.05). Only the ABCB1 rs1045642 CT and TT genotypes had a statistically significant correlation with EFS (<0.05, <0.05, respectively).

Conclusion: Our findings showed that genetic polymorphism could be associated with plasma MTX levels, toxicity experienced and EFS in Iranian pediatric ALL.
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http://dx.doi.org/10.22038/ijbms.2020.41754.9858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351433PMC
June 2020

ROR1 Is Expressed in Diffuse Large B-Cell Lymphoma (DLBCL) and a Small Molecule Inhibitor of ROR1 (KAN0441571C) Induced Apoptosis of Lymphoma Cells.

Biomedicines 2020 Jun 23;8(6). Epub 2020 Jun 23.

Department of Oncology-Pathology, BioClinicum, Karolinska University Hospital Solna and Karolinska Institutet, 17164 Stockholm, Sweden.

The receptor tyrosine kinase ROR1 is absent in most normal adult tissues, but overexpressed in several malignancies. In this study, we explored clinical and functional inhibitory aspects of ROR1 in diffuse large B-cell lymphoma (DLBCL). ROR1 expression in tumor cells was more often observed in primary refractory DLBCL, Richter's syndrome and transformed follicular lymphoma than in relapsed and non-relapsed DLBCL patients ( < 0.001). A survival effect of ROR1 expression was preliminarily observed in relapsed/refractory patients independent of gender and stage but not of age, cell of origin and international prognostic index. A second generation small molecule ROR1 inhibitor (KAN0441571C) induced apoptosis of ROR1+ DLBCL cell lines, similar to venetoclax (BCL-2 inhibitor) but superior to ibrutinib (BTK inhibitor). The combination of KAN0441571C and venetoclax at EC concentrations induced almost complete killing of DLBCL cell lines. Apoptosis was accompanied by the downregulation of BCL-2 and MCL-1 and confirmed by the cleavage of PARP and caspases 3, 8, 9. PI3Kδ/AKT/mTOR (non-canonical Wnt pathway) as well as β-catenin and CK1δ (canonical pathway) were inactivated. In zebra fishes transplanted with a ROR1+ DLBCL cell line, KAN0441571C induced a significant tumor reduction. New drugs with mechanisms of action other than those available for DLBCL are warranted. ROR1 inhibitors might represent a novel promising approach.
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http://dx.doi.org/10.3390/biomedicines8060170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344684PMC
June 2020

A receptor tyrosine kinase ROR1 inhibitor (KAN0439834) induced significant apoptosis of pancreatic cells which was enhanced by erlotinib and ibrutinib.

PLoS One 2018 1;13(6):e0198038. Epub 2018 Jun 1.

Department of Oncology-Pathology, Immune and Gene therapy Lab, Cancer Center Karolinska (CCK), Karolinska University Hospital Solna, Stockholm, Sweden.

There is a great unmet medical need in pancreatic carcinoma (PC) for novel drugs with other mechanisms of action than existing. PC cells express the onco-fetal RTK ROR1, absent on most normal post-partem cells. ROR1 is involved in proliferation, survival, EMT and metastasis of tumor cells in various malignancies. A small molecule inhibitor (KAN0439834) (530 Da) targeting the TK domain of ROR1 was developed and the activity in ROR1 expressing human PC cell lines (n = 8) evaluated. The effects were compared to a murine mAb against the external part of ROR1, gemcitabine, erlotinib and ibrutinib. KAN0439834 induced significant apoptosis of the tumor cells. EC50 values for KAN0439834 varied between 250-650 nM depending on the cell line. The corresponding values for erlotinib and ibrutinib were 10-40 folds higher. KAN0439834 was much more effective in inducing tumor cell death than the ROR1 mAb although both inhibited ROR1 phosphorylation and downstream non-canonical Wnt pathway molecules. Combination of KAN0439834 with erlotinib or ibrutinib had significant additive effects on tumor cell death. A first-in-class small molecule ROR1 inhibitor (KAN0439834) showed promising in vitro activity against a number of human PC cell lines. Interesting is the additive effects of erlotinib and ibrutinib which warrants further studies as both these agents are in clinical trials for pancreatic carcinoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198038PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983484PMC
December 2018

Lenalidomide as immune adjuvant to a dendritic cell vaccine in chronic lymphocytic leukemia patients.

Eur J Haematol 2018 Jul 22;101(1):68-77. Epub 2018 May 22.

Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.

Objectives: We previously showed that immunization with ex vivo- generated autologous dendritic cells loaded with apoptotic tumor cells (Apo-DC) potentiated tumor-specific immunity in chronic lymphocytic leukemia (CLL) patients. Here, we evaluated safety and immunogenicity of Apo-DC in combination with lenalidomide, granulocyte-macrophage colony-stimulating factor (GM-CSF), and low-dose cyclophosphamide (CTX).

Methods: Ten previously untreated patients with slowly progressing CLL received 5 Apo-DC vaccinations and lenalidomide orally for 24 weeks either alone (cohort I, n = 5) or together with subcutaneous GM-CSF and intravenous CTX (cohort II, n = 5). Tumor-specific T-cell responses were measured by proliferation and IFN-γ ELISPOT assays. Immune monitoring was performed by flow cytometry.

Results: Dose-limiting toxicity was observed in 3/10 patients, 2 in cohort I and one in cohort II. One patient developed autoimmune hemolytic anemia and another grade 4 thrombocytopenia. Vaccine-induced immune responses were seen in 5/5 and 4/5 patients in cohort I and II, respectively. The expression of immune checkpoints on T cells did not change significantly.

Conclusions: Lenalidomide alone or in combination with GM-CSF and low-dose CTX as immune adjuvant to the Apo-DC vaccine elicited tumor-specific T-cell responses in CLL patients. However, unexpected toxicity was observed and caution is suggested in further exploring this drug as immune adjuvant in CLL.
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http://dx.doi.org/10.1111/ejh.13065DOI Listing
July 2018

Autologous T cells expressing the oncogenic transcription factor KLF6-SV1 prevent apoptosis of chronic lymphocytic leukemia cells.

PLoS One 2018 12;13(2):e0192839. Epub 2018 Feb 12.

Immune and Gene Therapy Laboratory, Cancer Centre Karolinska, Department of Oncology and Pathology, Karolinska Institute, Stockholm, Sweden.

Crosstalk between leukemic cells and the tumor microenvironment is of importance in chronic lymphocytic leukemia (CLL). T cells seem to sustain the survival of CLL cells by various mechanisms. The Krüppel-like family of transcription factors (KLFs) are identified as regulators of proliferation and cell death. In the present study, we analyzed the expression of the wild type (WT) gene KLF6 and the oncogenic splice variant 1 (KLF6-SV1) at the mRNA level in subsets of T cells from CLL patients (n = 29), multiple myeloma patients (n = 6) and normal donors (n = 10). RNA Silencing was used for wtKLF6 and KLF6-SV1. Tumor cell apoptosis was measured. A significant overexpression of wtKLF6 and KLF6-SV1 in T cells of CLL patients compared to normal donors and myeloma patients was noted (p<0.002). Western blot showed that both wtKLF6 and KLF6-SV1 were expressed in purified T cells from CLL patients. KLF6-SV1 siRNA transfection induced a significant down-regulation of KLF6-SV1 in CLL T cells, which lost the capability to sustain the growth of leukemic cells. However, no such a significant effect was seen after wtKLF6 transfection of the autologous T cells. The results suggest that KLF6-SV1 may play a role in the regulation of survival CLL cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192839PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809069PMC
April 2018

European Perspective on Biosimilars.

Authors:
Håkan Mellstedt

J Oncol Pract 2017 09;13(9_suppl):15s-16s

Karolinska Institute, Solna, Stockholm, Sweden.

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http://dx.doi.org/10.1200/JOP.2017.025569DOI Listing
September 2017

Glioblastoma-synthesized G-CSF and GM-CSF contribute to growth and immunosuppression: Potential therapeutic benefit from dapsone, fenofibrate, and ribavirin.

Tumour Biol 2017 May;39(5):1010428317699797

6 Department of Neurosurgery, University of Ulm, Ulm, Germany.

Increased ratio of circulating neutrophils to lymphocytes is a common finding in glioblastoma and other cancers. Data reviewed establish that any damage to brain tissue tends to cause an increase in G-CSF and/or GM-CSF (G(M)-CSF) synthesized by the brain. Glioblastoma cells themselves also synthesize G(M)-CSF. G(M)-CSF synthesized by brain due to damage by a growing tumor and by the tumor itself stimulates bone marrow to shift hematopoiesis toward granulocytic lineages away from lymphocytic lineages. This shift is immunosuppressive and generates the relative lymphopenia characteristic of glioblastoma. Any trauma to brain-be it blunt, sharp, ischemic, infectious, cytotoxic, tumor encroachment, or radiation-increases brain synthesis of G(M)-CSF. G(M)-CSF are growth and motility enhancing factors for glioblastomas. High levels of G(M)-CSF contribute to the characteristic neutrophilia and lymphopenia of glioblastoma. Hematopoietic bone marrow becomes entrained with, directed by, and contributes to glioblastoma pathology. The antibiotic dapsone, the lipid-lowering agent fenofibrate, and the antiviral drug ribavirin are Food and Drug Administration- and European Medicines Agency-approved medicines that have potential to lower synthesis or effects of G(M)-CSF and thus deprive a glioblastoma of some of the growth promoting contributions of bone marrow and G(M)-CSF.
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http://dx.doi.org/10.1177/1010428317699797DOI Listing
May 2017

Clinical and Immune Effects of Lenalidomide in Combination with Gemcitabine in Patients with Advanced Pancreatic Cancer.

PLoS One 2017 18;12(1):e0169736. Epub 2017 Jan 18.

Department of Oncology-Pathology, Cancer Center Karolinska (CCK), Karolinska Institutet, Stockholm, Sweden.

Purpose: To assess the immunomodulatory and clinical effects of lenalidomide with standard treatment of gemcitabine in patients with advanced pancreatic cancer.

Patients And Methods: Patients with advanced pancreatic cancer were treated in first line with lenalidomide orally for 21 days of a 28 days cycle and the standard regimen for gemcitabine. In Part I, which we previously have reported, the dose of lenalidomide was defined (n = 12). In Part II, every other consecutive patient was treated with either lenalidomide (Group A, n = 11) or gemcitabine (Group B, n = 10) during cycle 1. From cycle 2 on, all Part II patients received the combination.

Results: A significant decrease in the proliferative response of peripheral blood mononuclear cells and the frequency of DCs were noted in patients at baseline compared to healthy control donors while the frequencies of CD4+ and CD8+ T cells, NK-cells and MDSCs were significantly higher in patients compared to controls. In Group A, a significant increase in the absolute numbers of activated (HLA-DR+) CD4 and CD8 T cells and CD8 effector memory T cells (p<0.01) was noted during treatment. A statistical increment in the absolute numbers of Tregs were seen after cycle 1 (p<0.05). The addition of gemcitabine, reduced most lymphocyte subsets (p<0.05). In Group B, the proportion of lymphocytes remained unchanged during the study period. There was no difference in overall survival, progression free survival and survival rate at one year comparing the two groups.

Discussion: Patients with advanced pancreatic carcinoma had impaired immune functions. Lenalidomide augmented T cell reactivities, which were abrogated by gemcitabine. However, addition of lenalidomide to gemcitabine seemed to have no therapeutic impact compared to gemcitabine alone in this non-randomized study.

Trial Registration: ClinicalTrials.gov NCT01547260.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169736PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242484PMC
August 2017

T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers.

Haematologica 2017 03 7;102(3):562-572. Epub 2016 Dec 7.

Immune and Gene Therapy Laboratory, Department of Oncology & Pathology, Cancer Centre Karolinska, Karolinska Institutet, Stockholm, Sweden.

Chronic lymphocytic leukemia is characterized by impaired immune functions largely due to profound T-cell defects. T-cell functions also depend on co-signaling receptors, inhibitory or stimulatory, known as immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1). Here we analyzed the T-cell phenotype focusing on immune checkpoints and activation markers in chronic lymphocytic leukemia patients (n=80) with different clinical characteristics and compared them to healthy controls. In general, patients had higher absolute numbers of CD3 cells and the CD8 subset was particularly expanded in previously treated patients. Progressive patients had higher numbers of CD4 and CD8 cells expressing PD-1 compared to healthy controls, which was more pronounced in previously treated patients (=0.0003 and =0.001, respectively). A significant increase in antigen-experienced T cells was observed in patients within both the CD4 and CD8 subsets, with a significantly higher PD-1 expression. Higher numbers of CD4 and CD8 cells with intracellular CTLA-4 were observed in patients, as well as high numbers of proliferating (Ki67) and activated (CD69) CD4 and CD8 cells, more pronounced in patients with active disease. The numbers of Th1, Th2, Th17 and regulatory T cells were substantially increased in patients compared to controls (<0.05), albeit decreasing to low levels in pre-treated patients. In conclusion, chronic lymphocytic leukemia T cells display increased expression of immune checkpoints, abnormal subset distribution, and a higher proportion of proliferating cells compared to healthy T cells. Disease activity and previous treatment shape the T-cell profile of chronic lymphocytic leukemia patients in different ways.
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http://dx.doi.org/10.3324/haematol.2016.151100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394965PMC
March 2017

Phase I-II study of lenalidomide and alemtuzumab in refractory chronic lymphocytic leukemia (CLL): effects on T cells and immune checkpoints.

Cancer Immunol Immunother 2017 01 4;66(1):91-102. Epub 2016 Nov 4.

Department of Hematology, Karolinska University Hospital Solna, 171 76, Stockholm, Sweden.

This phase I-II study explored safety, immunomodulatory and clinical effects of lenalidomide (weeks 1-16) and alemtuzumab (weeks 5-16) in 23 patients with refractory chronic lymphocytic leukemia. Most patients had Rai stage III/IV disease and were heavily pretreated (median 4 prior therapies), and 61% had del(17p)/del(11q). Eleven of 19 evaluable patients (58%) responded, with a median response duration of 12 months (1-29+); time to progression was short in non-responders. Lenalidomide had a narrow therapeutic dose range, 2.5 mg/day was not efficient, and maximum tolerated dose was 5 mg/day. Grade 3-4 neutropenia and thrombocytopenia occurred in 84 and 55%, 30% had febrile neutropenia, and CMV-reactivation requiring valganciclovir occurred in 30% of patients. The frequency of proliferating (Ki67) CD8 T cells was increased at week 4, with further increase in both the CD4 and CD8 subsets (p < 0.01 and <0.05), which was accompanied by significant upregulation of HLA-DR after addition of alemtuzumab. Antigen-experienced cells increased at week 4 as the frequency of effector memory cells increased in the CD8 subset (p < 0.003), while effector cells decreased in both the CD8 and CD4 subsets (p < 0.0001 and p < 0.01). The Th1/Th2 balance was unchanged at week 4 but shifted toward a Th2 profile after combination therapy. At end of treatment, the frequency of Th17 and regulatory T cells was reduced (p < 0.01), naïve T cells decreased, and effector memory T cells increased (p < 0.05 and p < 0.01). Granzyme B T cells increased at 30-week follow-up (p < 0.05). PD-1 expression was unaffected. In conclusion, low-dose lenalidomide and alemtuzumab induced major perturbations of T cells, including increased proliferative activity and cytotoxic potential.
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http://dx.doi.org/10.1007/s00262-016-1922-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222940PMC
January 2017

Dishevelled proteins are significantly upregulated in chronic lymphocytic leukaemia.

Tumour Biol 2016 Sep 16;37(9):11947-11957. Epub 2016 Apr 16.

Department of Oncology-Pathology, Immune and Gene Therapy Lab, Cancer Center Karolinska (CCK), Karolinska University Hospital Solna and Karolinska Institutet, Stockholm, Sweden.

Dishevelled (DVL) proteins are components of the Wnt signalling pathways, and increased expression is associated with various malignancies. Information on DVLs in chronic lymphatic leukaemia (CLL) is limited. The aim of the present study was to investigate the role of DVLs in CLL cells and association with Wnt pathways downstream of ROR1. DVL1, 2 and 3 were exclusively expressed in CLL cells as compared to normal peripheral blood mononuclear cells (PBMCs). The expression of DVL1 and DVL3 proteins was significantly more pronounced in progressive than in non-progressive disease (p < 0.01), whereas the level of DVL2 was significantly higher in non-progressive as compared to progressive disease (p < 0.001). Treatment of CLL cells with anti-ROR1 specific monoclonal antibodies induced dephosphorylation of ROR1 as well as of tyrosine and serine residues of both DVL2 and DVL3. However, gene silencing of DVLs in the CLL cell line (EHEB) did not induce detectable apoptosis. Non-progressive CLL patients had a different protein activity pattern with regard to Wnt signalling pathway proteins as GSK-3β, β-catenin and AKT as compared to progressive disease. The DVL2 protein may play a role in the activation of signalling pathways in CLL during early stages of the disease, while DVL1 and 3 may have a role in later phases of the leukaemia.
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http://dx.doi.org/10.1007/s13277-016-5039-5DOI Listing
September 2016

Differential expression of viral agents in lymphoma tissues of patients with ABC diffuse large B-cell lymphoma from high and low endemic infectious disease regions.

Oncol Lett 2016 Oct 16;12(4):2782-2788. Epub 2016 Aug 16.

Department of Oncology and Pathology, Karolinska Institute, 171 77 Stockholm, Sweden.

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin's lymphoma (NHL) in adults, accounts for approximately 30-40% of newly diagnosed lymphomas worldwide. Environmental factors, such as viruses and bacteria, may contribute to cancer development through chronic inflammation and the integration of oncogenes, and have previously been indicated in cervical cancer, hepatocellular carcinoma, gastric cancer and lymphoproliferative disorders. In the present study, the presence of microbial agents was analyzed in the lymphoma tissue of patients with activated B-cell like (ABC) DLBCL. The present study compared two groups of patients from geographically varied regions that possess a difference in the prevalence of viral and other microbial agents. The patient populations were from Sweden (a low endemic infectious disease region) and Egypt (a high endemic infectious disease region). A differential expression of several viruses in lymphoma tissues was noted when comparing Swedish and Egyptian patients. JC polyomavirus (JCV) was detected in Swedish and Egyptian patients and, uniquely, the complete hepatitis B virus (HBV) genome was detected only in Egyptian lymphoma patients. None of these viruses were detected in control lymph tissues from Sweden or Egypt. In total, 38% of the Egyptian patients were found to have HBV surface antigens (HBsAgs) in their serum; however, HBsAgs were not found in any of the Swedish patients. The percentage of serum HBsAgs in Egyptian patients with ABC DLBCL was significantly increased compared with the general Egyptian population (P<0.05). The present study may support a notion that viral agents, including JCV and HBV, may be involved in the tumorigenesis of DLBCL in regions of high infectious disease.
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http://dx.doi.org/10.3892/ol.2016.5012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038175PMC
October 2016

Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia.

PLoS One 2015 12;10(11):e0142310. Epub 2015 Nov 12.

Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Background: ROR1 is a receptor tyrosine kinase expressed in chronic lymphocytic leukemia (CLL) and several other malignancies but absent in most adult normal tissues. ROR1 is considered an onco-fetal antigen. In the present study we analysed spontaneous humoral and cellular immunity against ROR1 in CLL patients.

Materials And Methods: Antibodies against ROR1 were analysed in 23 patients and 20 healthy donors by ELISA and Western blot. Purified serum IgG from patients was tested for cytotoxicity against CLL cells using the MTT viability assay. A cellular immune response against ROR1 derived HLA-A2 restricted 9 aa and 16 aa long peptides were analysed using peptide loaded dendritic cells co-cultured with autologous T cells from CLL patients (n = 9) and healthy donors (n = 6). IFN-γ, IL-5 and IL-17A-secreting T cells were assessed by ELISPOT and a proliferative response using a H3-thymidine incorporation assay.

Results: The majority of CLL patients had antibodies against ROR1. Significantly higher titers of anti-ROR1 antibodies were noted in patients with non-progressive as compared to progressive disease. The extracellular membrane-close ROR1 KNG domain seemed to be an immunodominant epitope. Ten patients with high titers of anti-ROR1 binding antibodies were tested for cytotoxicity. Five of those had cytotoxic anti-ROR1 antibodies against CLL cells. ROR1-specific IFN-γ and IL-17A producing T cells could be detected in CLL patients, preferentially in non-progressive as compared to patients with progressive disease (p<0.05).

Conclusion: ROR1 seemed to spontaneously induce a humoral as well as a T cell response in CLL patients. The data support the notion that ROR1 might be a specific neo-antigen and may serve as a target for immunotherapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142310PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642968PMC
June 2016

Biosimilars - terms of use.

Curr Med Res Opin 2015 Dec 27;31(12):2325-30. Epub 2015 Oct 27.

c c Istituto Clinico Humanitas - IRCCS in Gastroenterology , Milan , Italy.

The impending expiry of the patent on a number of leading biologic drugs has led to a surge in the development of 'biosimilar' or 'follow-on' products. However, in contrast to generic small-molecule medicines, biosimilars are not identical to their reference products. The differences and complexities surrounding both the molecular structure and the manufacturing process for biologics and biosimilars have resulted in a lack of clarity regarding the terms used in different parts of the world to define various aspects of development and utilization such as regulatory approval, pharmacovigilance, interchangeability and treatment-naivety. This makes quantitative evaluation of biosimilars a great challenge to both the scientific community and regulatory agencies. This manuscript attempts to clarify the terms used and address an important knowledge gap which is currently resulting in an increasing rush to position biosimilars for certain indications and patients in the absence of agreed upon definitions.
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http://dx.doi.org/10.1185/03007995.2015.1098601DOI Listing
December 2015

Ibrutinib-A double-edge sword in cancer and autoimmune disorders.

J Drug Target 2016 11;24(5):373-85. Epub 2015 Sep 11.

b Department of Oncology-Pathology, Immune and Gene therapy Lab , Cancer Center Karolinska (CCK), Karolinska University Hospital Solna and Karolinska Institute , Stockholm , Sweden .

Targeted therapies have appeared as new treatment options for several disease types, including cancer and autoimmune disorders. Of several targets, tyrosine kinases (TKs) are among the most promising. Overexpression of TKs provides a target for novel therapeutic agents, including small molecule inhibitors of tyrosine kinases (TKI). Ibrutinib (PCI-32765) is a TKI of Bruton's tyrosine kinase (Btk), a key kinase of the B-cell receptor signaling pathway that plays a significant role in the proliferation, differentiation and survival of B cells. In addition to inhibitory effects, recent studies have shown that ibrutinib has multiple immunomodulatory effects. It binds covalently to IL-2 inducible tyrosine kinase (Itk) in T lymphocytes and suppresses the survival of T-helper (Th) 2 cells. This changes the balance of Th1/Th2 cells toward Th1 subset, which are the main immune cells targeting tumor cells. The dual activity of ibrutinib has paid a great attention and several studies are evaluating the anti-tumor and immunomodulatory effects in cancer, autoimmune disorders and infectious diseases. In this article we review the inhibitory and immunomodulatory effects of ibrutinib in B-cell malignancies, autoimmune diseases and infections, as well as the communication between the Ror1 receptor tyrosine kinase and BCR and effects of ibrutinib on this crosstalk.
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http://dx.doi.org/10.3109/1061186X.2015.1086357DOI Listing
December 2016

Dendritic cell regulation of NK-cell responses involves lymphotoxin-α, IL-12, and TGF-β.

Eur J Immunol 2015 Jun 17;45(6):1783-93. Epub 2015 Apr 17.

Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.

Dendritic cell (DC) vaccines induce T-cell responses in cancer patients. However, there is a paucity of data regarding the role of DC vaccines in shaping natural killer (NK) cell responses. Here, we observe that NK cells are less activated following DC vaccination. In vitro, DC-mediated inhibition of NK cells did not require cell-to-cell contact, but required increased Signal transducer and activator of transcription 3 (STAT3) phosphorylation (pSTAT3) in DCs. When phosphorylation of STAT3 was inhibited in DCs, we found that DCs did not suppress NK cells, and observed an increase in the production of lymphotoxin-alpha (LTα) and interleukin-12 (IL-12) as well as reduced release of transforming growth factor beta (TGF-β). The addition of recombinant LTα or IL-12 to the DC-NK-cell cocultures restored NK-cell activity, and neutralization of TGF-β resulted in elevated production of LTα and IL-12 from DCs. Compared with LPS, DCs matured with a cocktail of R848, poly I:C, and IFN-γ showed reduced levels of pSTAT3 and higher levels of LTα and IL-12 and did not inhibit NK-cell activity. These results show that LTα, IL-12, and TGF-β are involved in the cross-talk between NK cells and DCs. Our findings have important implications for the development of DC-based vaccination strategies to potentiate NK-cell responses in patients with cancer.
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http://dx.doi.org/10.1002/eji.201444885DOI Listing
June 2015

Mucin 1-specific active cancer immunotherapy with tecemotide (L-BLP25) in patients with multiple myeloma: an exploratory study.

Hum Vaccin Immunother 2014 ;10(11):3394-408

a Karolinska Institute and Karolinska University Hospital ; Stockholm , Sweden.

Patients (n = 34) with previously untreated, slowly progressive asymptomatic stage I/II multiple myeloma or with stage II/III multiple myeloma in stable response/plateau phase following conventional anti-tumor therapy were immunized repeatedly with the antigen-specific cancer immunotherapeutic agent tecemotide (L-BLP25). Additionally, patients were randomly allocated to either single or multiple low doses of cyclophosphamide to inhibit regulatory T cells (Treg). Immunization with tecemotide resulted in the induction/augmentation of a mucin 1-specific immune response in 47% of patients. The immune responses appeared to involve a Th1-like cellular immune response involving CD4 and CD8 T cells. The rate of immune responses was similar with single versus multiple dosing of cyclophosphamide and in patients with vs. without pre-existing mucin 1 immunity. On-treatment reductions in the slope of M-protein concentration over time (but not fulfilling clinical criteria for responses with conventional anti-tumor agents) were observed in 45% of evaluable patients, predominantly in those without versus with pre-existing mucin 1 immunity and in patients with early stage disease. No differences were seen in patients receiving single or multiple cyclophosphamide dosing. Treatment with tecemotide was generally well tolerated. Repeated vs. single dosing of cyclophosphamide had no impact on Treg numbers and was stopped after a case of fatal encephalitis that was assessed as possibly study-related. Tecemotide immunotherapy induces mucin 1-specific cellular immune responses in a substantial proportion of patients, with preliminary evidence of changes in the M-protein concentration time curve in a subset of patients.
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http://dx.doi.org/10.4161/hv.29918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514088PMC
October 2015

Awareness and understanding of cancer immunotherapy in Europe.

Hum Vaccin Immunother 2014 ;10(7):1828-35

a Karolinska Institute; Stockholm, Sweden.

The use of immunotherapy in the management of cancer is growing, and a range of new immunotherapeutic strategies is becoming available. It is important that people involved in the care of cancer understand how cancer immunotherapies differ from conventional chemotherapy and apply this knowledge to their clinical practice. Therefore, from August-September 2011 we undertook a survey of awareness, attitudes, and perceptions of cancer immunotherapy among 426 healthcare professionals (HCPs) in Europe with the aim of identifying and prioritizing educational needs. Nearly all (98%) HCPs were aware of cancer immunotherapy. While 68% of HCPs indicated a high level of interest in cancer immunotherapies, only 24% of the HCPs had direct experience with them. Overall perceptions of cancer immunotherapy among HCPs were largely positive (60%) and rarely negative (3%). The key advantages of cancer immunotherapy were perceived to be good safety and tolerability (75%), a targeted mechanism of action (61%) and good efficacy (48%). The leading barriers to use of immunotherapies were costs of treatment (58%), past clinical trial failures (45%), and access/formulary restrictions (44%). The results indicate that, among the respondents, awareness of cancer immunotherapy was high but that knowledge levels varied and direct experience with their use was limited. There appears to be a need for educational activities on cancer immunotherapy, as well as generation and communication of clinical data on long-term efficacy and safety.
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http://dx.doi.org/10.4161/hv.28943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4186017PMC
July 2015

The PI3K/AKT/mTOR pathway is involved in direct apoptosis of CLL cells induced by ROR1 monoclonal antibodies.

Br J Haematol 2015 May 19;169(3):455-8. Epub 2014 Nov 19.

Department of Oncology-Pathology, Immune and Gene Therapy Lab, CCK, Karolinska Institute and Karolinska University Hospital Solna, Stockholm, Sweden.

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http://dx.doi.org/10.1111/bjh.13228DOI Listing
May 2015

The receptor tyrosine kinase ROR1--an oncofetal antigen for targeted cancer therapy.

Semin Cancer Biol 2014 Dec 25;29:21-31. Epub 2014 Jul 25.

Department of Oncology-Pathology, Immune and Gene Therapy Lab, Cancer Center Karolinska (CCK), Karolinska University Hospital Solna and Karolinska Institutet, Stockholm, Sweden. Electronic address:

Targeted cancer therapies have emerged as new treatment options for various cancer types. Among targets, receptor tyrosine kinases (RTKs) are among the most promising. ROR1 is a transmembrane RTK of importance during the normal embryogenesis for the central nervous system, heart, lung and skeletal systems, but is not expressed in normal adult tissues. However, ROR1 is overexpressed in several human malignancies and may act as a survival factor for tumor cells. Its unique expression by malignant cells may provide a target for novel therapeutics including monoclonal antibodies (mAbs) and small molecule inhibitors of tyrosine kinases (TKI) for the treatment of cancer. Promising preclinical results have been reported in e.g. chronic lymphocytic leukemia, pancreatic carcinoma, lung and breast cancer. ROR1 might also be an interesting oncofetal antigen for active immunotherapy. In this review, we provide an overview of the ROR1 structure and functions in cancer and highlight emerging therapeutic options of interest for targeting ROR1 in tumor therapy.
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http://dx.doi.org/10.1016/j.semcancer.2014.07.005DOI Listing
December 2014

Overexpression of the epithelial cell adhesion molecule is associated with a more favorable prognosis and response to platinum-based chemotherapy in ovarian cancer.

J Gynecol Oncol 2014 Jul 3;25(3):221-8. Epub 2014 Jul 3.

Department of Gynecology, European Competence Center for Ovarian Cancer, Campus Virchow Klinikum, Charité-University Medicine of Berlin, Berlin, Germany.

Objective: Epithelial cell adhesion molecule (EpCAM) has experienced a renaissance lately as a binding site for targeted therapy as well as a prognostic marker in epithelial malignancies. Aim of this study was to study EpCAM as a potential prognostic marker in epithelial ovarian cancer (EOC).

Methods: EpCAM expression was assessed by immunohistochemistry on paraffin-embedded primary EOC-tissue samples. EpCAM overexpression was defined as an expression of EpCAM of 76% to 100%. Tissue samples and clinical data were systematically collected within the international and multicenter "Tumorbank Ovarian Cancer" network.

Results: Seventy-four patients, diagnosed with EOC between 1994 and 2009, were included in the study (median age, 56 years; range, 31 to 86 years). The majority of the patients (81.1%) presented with an advanced stage International Federation of Gynecology and Obstetrics (FIGO) III/IV disease. Histology was of the serous type in 41 patients (55.4%), endometrioid in 19 (25.6%), and mucinous in 14 (19%). EpCAM was overexpressed in 87.7%. Serous tumors overexpressed EpCAM significantly more often than mucinous tumors (87.8% vs. 78.6%, p=0.045); while no significant difference was noted between the other histological subgroups. EpCAM overexpression was significantly associated with a better progression free survival and higher response rates to platinum based chemotherapy (p=0.040 and p=0.048, respectively). EpCAM was identified as an independent prognostic marker for overall survival (p=0.022).

Conclusion: Our data indicate a significant association of EpCAM overexpression with a more favorable survival in EOC-patients. Serous cancers showed a significant EpCAM overexpression compared to mucinous types. Larger multicenter analyses are warranted to confirm these findings.
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http://dx.doi.org/10.3802/jgo.2014.25.3.221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102741PMC
July 2014

Therapeutic vaccines for cancer: an overview of clinical trials.

Nat Rev Clin Oncol 2014 Sep 8;11(9):509-24. Epub 2014 Jul 8.

Cancer Centre Karolinska, Department of Oncology, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.

The therapeutic potential of host-specific and tumour-specific immune responses is well recognized and, after many years, active immunotherapies directed at inducing or augmenting these responses are entering clinical practice. Antitumour immunization is a complex, multi-component task, and the optimal combinations of antigens, adjuvants, delivery vehicles and routes of administration are not yet identified. Active immunotherapy must also address the immunosuppressive and tolerogenic mechanisms deployed by tumours. This Review provides an overview of new results from clinical studies of therapeutic cancer vaccines directed against tumour-associated antigens and discusses their implications for the use of active immunotherapy.
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http://dx.doi.org/10.1038/nrclinonc.2014.111DOI Listing
September 2014

[Cheaper with biosimilars, but safety comes first].

Authors:
Håkan Mellstedt

Lakartidningen 2014 May 27-Jun 10;111(22-23):1011

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August 2014

Telomerase (GV1001) vaccination together with gemcitabine in advanced pancreatic cancer patients.

Int J Oncol 2014 Sep 11;45(3):1293-303. Epub 2014 Jun 11.

Department of Oncology-Pathology (Radiumhemmet), Karolinska Institutet and Karolinska University Hospital Solna, SE-17176 Stockholm, Sweden.

Telomerase is expressed in 85-90 % of pancreatic adenocarcinomas and might be a target for active cancer immunotherapy. A study was conducted to investigate safety and immunogenicity in non-resectable pancreatic carcinoma patients using a 16-amino acid telomerase peptide (GV1001) for vaccination in combination with GM-CSF and gemcitabine as first line treatment. Three different vaccine treatment schedules were used; [A (n=6), B (n=6) and C (n=5)]. Groups A/B received GV1001, GM-CSF and gemcitabine concurrently. Group C received initially GV1001 and GM-CSF while gemcitabine was added at disease progression. Group D (n=4) was treated with gemcitabine alone. Adverse events (AE) related to vaccination were mild (grades I-II). Grade III AEs were few and transient. An induced GV 1001‑specific immune response was defined as an increase ≥2 above the baseline value in one of the assays (DTH, proliferation, ELISPOT and cytokine secretion assays, respectively). A telomerase‑specific immune response was noted in 4/6 patients in group A, 4/6 patients in group B and 2/5 patients in group C. An induced ras‑specific immune response (antigenic spreading) was seen in 5 of the 17 patients. The cytokine pattern was that of a Th1-like profile. A treatment induced telomerase or ras response was also noted in group D. All responses were weak and transient. A significant decrease in regulatory T-cells over time was noted in patients in groups A and B (p<0.05). Telomerase vaccination (GV1001) in combination with chemotherapy appeared to be safe but the immune responses were weak and transient. Measures have to be taken to optimize immune responses of GV1001 for it to be considered of clinical interest.
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http://dx.doi.org/10.3892/ijo.2014.2496DOI Listing
September 2014

Opticin, a small leucine-rich proteoglycan, is uniquely expressed and translocated to the nucleus of chronic lymphocytic leukemia cells.

Exp Hematol Oncol 2013 Aug 28;2(1):23. Epub 2013 Aug 28.

Immune and Gene Therapy Lab, CCK, Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden.

Background: Opticin (OPTC) is a member of the small leucine-rich proteoglycan (SLRP) family and is localized particularly in certain extracellular matrices. We have previously reported the unique expression of another SLRP, fibromodulin (FMOD) in the leukemic cells of patients with chronic lymphocytic leukemia (CLL). OPTC is located in the same region as FMOD on chromosome 1 (1q32.1). Cluster up-regulation of genes may be observed in malignancies and the aim of the present study was to analyze the expression of OPTC in CLL cells.

Methods: The expression of OPTC was tested by RT-PCR and realtime qPCR in PBMC from CLL patients, other hematological malignancies and healthy controls. The presence of OPTC protein, and its subcellular localization, was investigated using fractionation methods where the obtained lysate fractions were analyzed by Western blotting. Deglycosylation experiments were performed to investigate the glycosylation status of the CLL OPTC.

Results: OPTC was expressed at the gene level in all patients with CLL (n = 90) and in 2/8 patients with mantle cell lymphoma (MCL) but not in blood mononuclear cells of healthy control donors (n = 20) or in tumor samples from nine other types of hematological malignancies. OPTC was detected by Western blot in all CLL samples analyzed (n = 30) but not in normal leukocytes (n = 10). Further analysis revealed a CLL-unique unglycosylated 37 kDa core protein that was found to be located preferentially in the cell nucleus and endoplasmic reticulum (ER) of the CLL cells.

Conclusions: A 37 kDa unglycosylated OPTC protein was detected in ER and in the nucleus of CLL cells and not in healthy control donors. The function of this OPTC core protein remains unclear but its CLL-specific expression and subcellular localization warrants further investigations in the pathobiology of CLL.
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http://dx.doi.org/10.1186/2162-3619-2-23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3766095PMC
August 2013

Clinical considerations for biosimilar antibodies.

Authors:
Håkan Mellstedt

EJC Suppl 2013 Dec;11(3):1-11

Professor of Oncological Biotherapy, Department of Oncology-Pathology, Karolinska Institute, SE-171 76 Stockholm, Sweden.

Biosimilar agents are approximate copies of branded biologic therapies. Since the first biosimilar was authorized in the European Union in 2006, fifteen additional agents have been approved by the European Medicines Agency, including two biosimilar monoclonal antibodies (mAbs). Biosimilar mAbs represent a distinct class given their large molecular size, complex protein structure, and post-translational modifications. While guidelines have been established for the development, approval, and use of biosimilars, further scrutiny and discussion is necessary to fully understand their potential impact on clinical outcomes. This review takes a critical look at the structural complexity of biosimilar mABs, the feasibility of indication extrapolation, the impact of product variability on immunogenicity, the importance of comprehensive pharmacovigilance, and the potential for ongoing pharmacoeconomic impact.
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http://dx.doi.org/10.1016/S1359-6349(13)70001-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048039PMC
December 2013
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