Publications by authors named "Johan Richter"

95 Publications

Optical Brain Biopsy with a Fluorescence and Vessel Tracing Probe.

Oper Neurosurg (Hagerstown) 2021 Jun 30. Epub 2021 Jun 30.

Department of Biomedical Engineering, Linköping University, Linköping, Sweden.

Background: Accurate stereotactic biopsies of brain tumors are imperative for diagnosis and tailoring of the therapy. Repetitive needle insertions enhance risks of brain lesioning, hemorrhage, and complications due to prolonged procedure.

Objective: To investigate clinical benefits of a combined 5-aminolaevulinic acid (5-ALA) fluorescence and laser Doppler flowmetry system for the detection of malignant brain tumor and blood vessels in stereotactic biopsies.

Methods: Planning of targets and trajectories was followed by optical measurements in 20 patients, using the Leksell Stereotactic System and a manual insertion device. Fluorescence spectra, microvascular blood flow, and tissue grayness were recorded each millimeter along the paths. Biopsies were taken at preplanned positions. The diagnoses were compared with the fluorescence signals. The recordings were plotted against measurement positions and compared. Sites indicating a risk of hemorrhage were counted as well as the time for the procedures.

Results: Signals were recorded along 28 trajectories, and 78 biopsies were collected. The final diagnosis showed 17 glioblastomas, 2 lymphomas, and 1 astrocytoma grade III. Fluorescence was seen along 23 of the paths with 4 having the peak of 5-ALA fluorescence 3 mm or more from the precalculated target. There was increased microcirculation in 40 of 905 measured positions. The measurement time for each trajectory was 5 to 10 min.

Conclusion: The probe provided direct feedback of increased blood flow along the trajectory and of malignant tissue in the vicinity of the target. The method can increase the precision and the safety of the biopsy procedure and reduce time.
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http://dx.doi.org/10.1093/ons/opab216DOI Listing
June 2021

Successful tyrosine kinase inhibitor discontinuation outside clinical trials - data from the population-based Swedish chronic myeloid leukaemia registry.

Br J Haematol 2021 Jun 30;193(5):915-921. Epub 2021 Mar 30.

Department of Hematology, Oncology and Radiation Physics, Skåne University Hospital, Lund, Sweden.

Clinical trials show that tyrosine kinase inhibitor (TKI) treatment can be discontinued in selected patients with chronic myeloid leukaemia (CML). Although updated CML guidelines support such procedure in clinical routine, data on TKI stopping outside clinical trials are limited. In this retrospective study utilising the Swedish CML registry, we examined TKI discontinuation in a population-based setting. Out of 584 patients diagnosed with chronic-phase CML (CML-CP) in 2007-2012, 548 had evaluable information on TKI discontinuation. With a median follow-up of nine years from diagnosis, 128 (23%) discontinued TKI therapy (≥1 month) due to achieving a DMR (deep molecular response) and 107 (20%) due to other causes (adverse events, allogeneic stem cell transplant, pregnancy, etc). Among those stopping in DMR, 49% re-initiated TKI treatment (median time to restart 4·8 months). In all, 38 patients stopped TKI within a clinical study and 90 outside a study. After 24 months 41·1% of patients discontinuing outside a study had re-initiated TKI treatment. TKI treatment duration pre-stop was longer and proportion treated with second-generation TKI slightly higher outside studies, conceivably affecting the clinical outcome. In summary we show that TKI discontinuation in CML in clinical practice is common and feasible and may be just as successful as when performed within a clinical trial.
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http://dx.doi.org/10.1111/bjh.17392DOI Listing
June 2021

Gene therapy for infantile malignant osteopetrosis: review of pre-clinical research and proof-of-concept for phenotypic reversal.

Mol Ther Methods Clin Dev 2021 Mar 25;20:389-397. Epub 2020 Dec 25.

Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund University, Lund, Sweden.

Infantile malignant osteopetrosis is a devastating disorder of early childhood that is frequently fatal and for which there are only limited therapeutic options. Gene therapy utilizing autologous hematopoietic stem and progenitor cells represents a potentially advantageous therapeutic alternative for this multisystemic disease. Gene therapy can be performed relatively rapidly following diagnosis, will not result in graft versus host disease, and may also have potential for reduced incidences of other transplant-related complications. In this review, we have summarized the past sixteen years of research aimed at developing a gene therapy for infantile malignant osteopetrosis; these efforts have culminated in the first clinical trial employing lentiviral-mediated delivery of in autologous hematopoietic stem and progenitor cells.
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http://dx.doi.org/10.1016/j.omtm.2020.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848732PMC
March 2021

Experiences of awake surgery in non-tumoural epilepsy in eloquent localizations.

Clin Neurol Neurosurg 2020 12 25;199:106251. Epub 2020 Sep 25.

Department of Neurology, Linköping University Hospital, Region Östergötland, Sweden; Department of Biomedical and Clinical Sciences, Faculty of Medicine and Health Sciences Linköping University, Linköping, Sweden. Electronic address:

Background: Whilst modern awake intraoperative mapping has been widely accepted and implemented in the last decades in neuro-oncology, sparse reports have been published on the safety and efficiency of this approach in epilepsy surgery.

Method: This article reports four cases with different locations of epileptogenic zones as examples of possible safe and efficient resections.

Result: The results of the resections on seizure control were Engel 1 (no disabling seizures) in all cases and no patient experienced significant neurological deficits.

Discussion: The discussion focuses on aspects of the future of epilepsy surgery in a hodotopical paradigm.
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http://dx.doi.org/10.1016/j.clineuro.2020.106251DOI Listing
December 2020

Generation of gene-corrected functional osteoclasts from osteopetrotic induced pluripotent stem cells.

Stem Cell Res Ther 2020 05 15;11(1):179. Epub 2020 May 15.

Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund University, BMC A12, 221 84, Lund, Sweden.

Background: Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by non-functional osteoclasts and a fatal outcome early in childhood. About 50% of patients have mutations in the TCIRG1 gene.

Methods: IMO iPSCs were generated from a patient carrying a homozygous c.11279G>A (IVS18+1) mutation in TCIRG1 and transduced with a lentiviral vector expressing human TCIRG1. Embryoid bodies were generated and differentiated into monocytes. Non-adherent cells were harvested and further differentiated into osteoclasts on bovine bone slices.

Results: Release of the bone resorption biomarker CTX-I into the media of gene-corrected osteoclasts was 5-fold higher than that of the uncorrected osteoclasts and 35% of that of control osteoclasts. Bone resorption potential was confirmed by the presence of pits on the bones cultured with gene-corrected osteoclasts, absent in the uncorrected IMO osteoclasts.

Conclusions: The disease phenotype was partially corrected in vitro, providing a valuable resource for therapy development for this form of severe osteopetrosis.
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http://dx.doi.org/10.1186/s13287-020-01701-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227215PMC
May 2020

Plasma proteomics of biomarkers for inflammation or cancer cannot predict relapse in chronic myeloid leukaemia patients stopping tyrosine kinase inhibitor therapy.

Leuk Res 2020 03 23;90:106310. Epub 2020 Jan 23.

Department of Medical Sciences, Uppsala University, Uppsala, Sweden; Section of Hematology, Uppsala University Hospital, Uppsala, Sweden.

Several studies have now shown that chronic myeloid leukaemia (CML) patients in deep molecular remission may discontinue tyrosine kinase inhibitor (TKI) treatment with a treatment free remission (TFR) rate of approximately 40-60 %. Some factors influencing the possibility of TFR have been described but better tools are needed for individual prediction of long-term TFR. Herein, two multiplex panels were utilised to analyse a total of 162 different plasma proteins from 56 patients included in the TKI stopping trial EURO-SKI (Saussele et al., 2018). The purpose was to identify possible plasma protein markers for prediction of successful TKI discontinuation and to evaluate effects of TKI discontinuation on plasma protein profiles. No protein biomarkers sampled before TKI discontinuation could separate relapse cases from non-relapse cases but some plasma proteins differed between patients who relapsed and those who remained in TFR when followed over time after TKI cessation. In conclusion, the plasma protein markers in this study could not predict relapse after TKI discontinuation but may be of use to understand the mechanisms involved in maintenance of TFR.
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http://dx.doi.org/10.1016/j.leukres.2020.106310DOI Listing
March 2020

Increased Risk of Chronic Myeloid Leukemia Following Gastric Conditions Indicating Infection: A Case-Control Study.

Cancer Epidemiol Biomarkers Prev 2020 01 16;29(1):151-156. Epub 2019 Oct 16.

Department of Medical Sciences, Unit of Hematology, Uppsala University, Uppsala, Sweden.

Background: On the basis of a previous report of increased chronic myeloid leukemia (CML) risk following peptic ulcer, we hypothesized that chronic infection could serve as a risk factor for CML.

Methods: In a population-based, retrospective case-control study, we used Swedish registry data on 980 patients with CML and 4,960 age- and sex-matched controls to investigate associations between markers of previous infection with and CML incidence.

Results: Previous diagnoses of dyspepsia, gastritis or peptic ulcers, as well as previous proton pump inhibitor (PPI) medication, were all associated with a significantly increased risk of CML (RRs, 1.5-2.0; = 0.0005-0.05). Meanwhile, neither inflammatory bowel disease nor intake of NSAIDs were associated with CML, indicating that it is not gastrointestinal ulcer or inflammation that influences risk.

Conclusions: The consistent associations suggest a shared background between gastric conditions and CML, and strengthen the case that could constitute this common risk factor.

Impact: As the etiology of CML is practically unknown, and could potentially be a therapeutic target, even this indirect evidence encourages further studies on the potential involvement of in CML etiology.
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http://dx.doi.org/10.1158/1055-9965.EPI-19-0758DOI Listing
January 2020

A high-content cytokine screen identifies myostatin propeptide as a positive regulator of primitive chronic myeloid leukemia cells.

Haematologica 2020 08 3;105(8):2095-2104. Epub 2019 Oct 3.

Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden

Aberrantly expressed cytokines in the bone marrow (BM) niche are increasingly recognized as critical mediators of survival and expansion of leukemic stem cells. To identify regulators of primitive chronic myeloid leukemia (CML) cells, we performed a high-content cytokine screen using primary CD34 CD38 chronic phase CML cells. Out of the 313 unique human cytokines evaluated, 11 were found to expand cell numbers ≥2-fold in a 7-day culture. Focusing on novel positive regulators of primitive CML cells, the myostatin antagonist myostatin propeptide gave the largest increase in cell expansion and was chosen for further studies. Herein, we demonstrate that myostatin propeptide expands primitive CML and normal BM cells, as shown by increased colony-forming capacity. For primary CML samples, retention of CD34-expression was also seen after culture. Furthermore, we show expression of by CML mesenchymal stromal cells, and that myostatin propeptide has a direct and instant effect on CML cells, independent of myostatin, by demonstrating binding of myostatin propeptide to the cell surface and increased phosphorylation of STAT5 and SMAD2/3. In summary, we identify myostatin propeptide as a novel positive regulator of primitive CML cells and corresponding normal hematopoietic cells.
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http://dx.doi.org/10.3324/haematol.2019.220434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395258PMC
August 2020

Immunological monitoring of newly diagnosed CML patients treated with bosutinib or imatinib first-line.

Oncoimmunology 2019;8(9):e1638210. Epub 2019 Jul 13.

Hematology Research Unit Helsinki, University of Helsinki and Department of Hematology, Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland.

Changes in the immune system induced by tyrosine kinase inhibitors (TKI) have been shown to positively correlate with therapy responses in chronic myeloid leukemia (CML). However, only a few longitudinal studies exist and no randomized comparisons between two TKIs have been reported. Therefore, we prospectively analyzed the immune system of newly diagnosed CML patients treated with imatinib (n = 20) or bosutinib (n = 13), that participated in the randomized BFORE trial (NCT02130557). Comprehensive immunophenotyping, plasma protein profiling, and functional assays to determine activation levels of T and NK cells were performed at diagnosis, 3, and 12 months after therapy start. All results were correlated with clinical parameters such as Sokal risk and BCR-ABL load measured according to IS%. At diagnosis, low Sokal risk CML patients had a higher frequency of cytotoxic cells (CD8 + T and NK cells), increased cytotoxic potential of NK cells and lower frequency of naïve and central memory CD4 + T cells. Further, soluble plasma protein profile divided patients into two distinct clusters with different disease burden at diagnosis. During treatment, BCR-ABL IS% correlated with immunological parameters such as plasma proteins, together with different memory subsets of CD4+ and CD8 + T cells. Interestingly, the proportion and cytotoxic potential of NK cells together with several soluble proteins increased during imatinib treatment. In contrast, no major immunological changes were observed during bosutinib treatment. In conclusion, imatinib and bosutinib were shown to have differential effects on the immune system in this randomized clinical trial. Increased number and function of NK cells were especially observed during imatinib therapy.
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http://dx.doi.org/10.1080/2162402X.2019.1638210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685516PMC
February 2021

Hematopoietic Stem Cell-Targeted Neonatal Gene Therapy with a Clinically Applicable Lentiviral Vector Corrects Osteopetrosis in Mice.

Hum Gene Ther 2019 11 3;30(11):1395-1404. Epub 2019 Jul 3.

Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund University, Lund, Sweden.

Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by nonfunctional osteoclasts. Approximately 50% of the patients have mutations in the gene, encoding for a subunit of the osteoclast proton pump. Gene therapy represents a potential alternative treatment to allogeneic stem cell transplantation for IMO. The mouse is a model of IMO characterized by a 1,500 bp deletion in the gene, severe osteopetrosis, and a life span of only 3 weeks. Here we show that the osteopetrotic phenotype in mice can be reversed by hematopoietic stem cell-targeted gene therapy with a clinically applicable lentiviral vector expressing a wild-type form of human under the mammalian promoter elongation factor 1α short (EFS-hT). c-kit fetal liver cells transduced with EFS-hT were transplanted into sublethally irradiated mice by temporal vein injection 1 day after birth. A total of 9 of 12 mice survived long term (19-25 weeks) with evidence of tooth eruption, uncharacteristic of mice. Splenocytes were harvested 19-25 weeks after transplantation and differentiated into osteoclasts on bone slices to assess resorption and on plastic to assess TCIRG1 protein expression. Vector-corrected osteoclasts showed human TCIRG1 expression by Western blot. CTX-I release relative to that mediated by -derived osteoclasts increased 8-239-fold. Resorption pits on bone slices were observed for osteoclasts derived from 7/9 surviving transplanted mice. Histopathology of the bones of surviving animals showed varying degrees of rescued phenotype, the majority with almost full correction. The average vector copy number per cell in the bone marrow was 1.8 ± 0.5. Overall, 75% of transplanted mice exhibited long-term survival and marked reversal of the osteopetrotic bone phenotype. These findings represent a significant step toward the clinical application of gene therapy for IMO.
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http://dx.doi.org/10.1089/hum.2019.047DOI Listing
November 2019

Allogeneic stem cell transplantation for chronic myeloid leukemia in the TKI era: population-based data from the Swedish CML registry.

Bone Marrow Transplant 2019 11 8;54(11):1764-1774. Epub 2019 Apr 8.

Department of Medical Sciences, Division of Hematology, Uppsala University Hospital, Uppsala, Sweden.

Two decades after the introduction of tyrosine kinase inhibitors (TKI), a sizeable portion of patients with chronic myeloid leukemia (CML) in chronic phase (CP) still undergo allogeneic stem cell transplantation (allo-HSCT). We investigated the indications for allo-HSCT, clinical outcome, management of relapse, and post-transplant TKI treatment in a population-based setting using the Swedish CML registry. Of 118 CML patients transplanted between 2002 and 2017, 56 (47.4%) received allo-HSCT in first CP, among whom TKI resistance was the most common transplant indication (62.5%). For patients diagnosed with CML in CP at <65 years of age, the cumulative probability of undergoing allo-HSCT within 5 years was 9.7%. Overall 5-year survival was 96.2%, 70.1% and 36.9% when transplanted in first CP, second or later CP, and in accelerated phase or blast crisis, respectively. Risk factors for relapse were EBMT score >2 and reduced intensity conditioning, and for death, CP > 2 at time point of allo-HSCT only. Non-relapse mortality for patients transplanted in CP was 11.6%. Our data indicate that allo-HSCT still constitutes a reasonable therapeutic option for patients with CML in first CP, especially those resistant to TKI treatment, providing high long-term survival and low non-relapse mortality.
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http://dx.doi.org/10.1038/s41409-019-0513-5DOI Listing
November 2019

Telomere shortening correlates with leukemic stem cell burden at diagnosis of chronic myeloid leukemia.

Blood Adv 2018 07;2(13):1572-1579

Department of Hematology, Oncology, Hemostaseology, and Stem Cell Transplantation, Medical Faculty, RWTH Aachen University, Aachen, Germany.

Telomere length (TL) in peripheral blood (PB) cells of patients with chronic myeloid leukemia (CML) has been shown to correlate with disease stage, prognostic scores, response to therapy, and disease progression. However, due to considerable genetic interindividual variability, TL varies substantially between individuals, limiting its use as a robust prognostic marker in individual patients. Here, we compared TL of BCR-ABL, nonleukemic CD34CD38 hematopoietic stem cells (HSC) in the bone marrow of CML patients at diagnosis to their individual BCR-ABL leukemic stem cell (LSC) counterparts. We observed significantly accelerated telomere shortening in LSC compared with nonleukemic HSC. Interestingly, the degree of LSC telomere shortening was found to correlate significantly with the leukemic clone size. To validate the diagnostic value of nonleukemic cells as internal controls and to rule out effects of tyrosine kinase inhibitor (TKI) treatment on these nontarget cells, we prospectively assessed TL in 134 PB samples collected in deep molecular remission after TKI treatment within the EURO-SKI study (NCT01596114). Here, no significant telomere shortening was observed in granulocytes compared with an age-adjusted control cohort. In conclusion, this study provides proof of principle for accelerated telomere shortening in LSC as opposed to HSC in CML patients at diagnosis. The fact that the degree of telomere shortening correlates with leukemic clone's size supports the use of TL in leukemic cells as a prognostic parameter pending prospective validation. TL in nonleukemic myeloid cells seems unaffected even by long-term TKI treatment arguing against a reduction of telomere-mediated replicative reserve in normal hematopoiesis under TKI treatment.
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http://dx.doi.org/10.1182/bloodadvances.2018017772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039661PMC
July 2018

5-ALA fluorescence and laser Doppler flowmetry for guidance in a stereotactic brain tumor biopsy.

Biomed Opt Express 2018 May 20;9(5):2284-2296. Epub 2018 Apr 20.

Department of Biomedical Engineering, Linköping University, Sweden.

A fiber optic probe was developed for guidance during stereotactic brain biopsy procedures to target tumor tissue and reduce the risk of hemorrhage. The probe was connected to a setup for the measurement of 5-aminolevulinic acid (5-ALA) induced fluorescence and microvascular blood flow. Along three stereotactic trajectories, fluorescence (n = 109) and laser Doppler flowmetry (LDF) (n = 144) measurements were done in millimeter increments. The recorded signals were compared to histopathology and radiology images. The median ratio of protoporphyrin IX (PpIX) fluorescence and autofluorescence (AF) in the tumor was considerably higher than the marginal zone (17.3 vs 0.9). The blood flow showed two high spots (3%) in total. The proposed setup allows simultaneous and real-time detection of tumor tissue and microvascular blood flow for tracking the vessels.
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http://dx.doi.org/10.1364/BOE.9.002284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946788PMC
May 2018

Discontinuation of tyrosine kinase inhibitor therapy in chronic myeloid leukaemia (EURO-SKI): a prespecified interim analysis of a prospective, multicentre, non-randomised, trial.

Lancet Oncol 2018 06 4;19(6):747-757. Epub 2018 May 4.

Bergonié Cancer Institute, Inserm Unit 916, University of Bordeaux, Bordeaux, France. Electronic address:

Background: Tyrosine kinase inhibitors (TKIs) have improved the survival of patients with chronic myeloid leukaemia. Many patients have deep molecular responses, a prerequisite for TKI therapy discontinuation. We aimed to define precise conditions for stopping treatment.

Methods: In this prospective, non-randomised trial, we enrolled patients with chronic myeloid leukaemia at 61 European centres in 11 countries. Eligible patients had chronic-phase chronic myeloid leukaemia, had received any TKI for at least 3 years (without treatment failure according to European LeukemiaNet [ELN] recommendations), and had a confirmed deep molecular response for at least 1 year. The primary endpoint was molecular relapse-free survival, defined by loss of major molecular response (MMR; >0·1% BCR-ABL1 on the International Scale) and assessed in all patients with at least one molecular result. Secondary endpoints were a prognostic analysis of factors affecting maintenance of MMR at 6 months in learning and validation samples and the cost impact of stopping TKI therapy. We considered loss of haematological response, progress to accelerated-phase chronic myeloid leukaemia, or blast crisis as serious adverse events. This study presents the results of the prespecified interim analysis, which was done after the 6-month molecular relapse-free survival status was known for 200 patients. The study is ongoing and is registered with ClinicalTrials.gov, number NCT01596114.

Findings: Between May 30, 2012, and Dec 3, 2014, we assessed 868 patients with chronic myeloid leukaemia for eligibility, of whom 758 were enrolled. Median follow-up of the 755 patients evaluable for molecular response was 27 months (IQR 21-34). Molecular relapse-free survival for these patients was 61% (95% CI 57-64) at 6 months and 50% (46-54) at 24 months. Of these 755 patients, 371 (49%) lost MMR after TKI discontinuation, four (1%) died while in MMR for reasons unrelated to chronic myeloid leukaemia (myocardial infarction, lung cancer, renal cancer, and heart failure), and 13 (2%) restarted TKI therapy while in MMR. A further six (1%) patients died in chronic-phase chronic myeloid leukaemia after loss of MMR and re-initiation of TKI therapy for reasons unrelated to chronic myeloid leukaemia, and two (<1%) patients lost MMR despite restarting TKI therapy. In the prognostic analysis in 405 patients who received imatinib as first-line treatment (learning sample), longer treatment duration (odds ratio [OR] per year 1·14 [95% CI 1·05-1·23]; p=0·0010) and longer deep molecular response durations (1·13 [1·04-1·23]; p=0·0032) were associated with increasing probability of MMR maintenance at 6 months. The OR for deep molecular response duration was replicated in the validation sample consisting of 171 patients treated with any TKI as first-line treatment, although the association was not significant (1·13 [0·98-1·29]; p=0·08). TKI discontinuation was associated with substantial cost savings (an estimated €22 million). No serious adverse events were reported.

Interpretation: Patients with chronic myeloid leukaemia who have achieved deep molecular responses have good molecular relapse-free survival. Such patients should be considered for TKI discontinuation, particularly those who have been in deep molecular response for a long time. Stopping treatment could spare patients from treatment-induced side-effects and reduce health expenditure.

Funding: ELN Foundation and France National Cancer Institute.
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http://dx.doi.org/10.1016/S1470-2045(18)30192-XDOI Listing
June 2018

Osteoclasts degrade bone and cartilage knee joint compartments through different resorption processes.

Arthritis Res Ther 2018 04 10;20(1):67. Epub 2018 Apr 10.

Nordic Bioscience, Herlev Hovedgade 205-207, 2730, Herlev, Denmark.

Background: Osteoclasts have been strongly implicated in osteoarthritic cartilage degradation, at least indirectly via bone resorption, and have been shown to degrade cartilage in vitro. The osteoclast resorption processes required to degrade subchondral bone and cartilage-the remodeling of which is important in the osteoarthritic disease process-have not been previously described, although cathepsin K has been indicated to participate. In this study we profile osteoclast-mediated degradation of bovine knee joint compartments in a novel in vitro model using biomarkers of extracellular matrix (ECM) degradation to assess the potential of osteoclast-derived resorption processes to degrade different knee joint compartments.

Methods: Mature human osteoclasts were cultured on ECMs isolated from bovine knees-articular cartilage, cortical bone, and osteochondral junction ECM (a subchondral bone-calcified cartilage mixture)-in the presence of inhibitors: the cystein protease inhibitor E-64, the matrix metalloproteinase (MMP) inhibitor GM6001, or the vacuolar-type H-ATPase (V-ATPase) inhibitor diphyllin. Biomarkers of bone (calcium and C-terminal type I collagen (CTX-I)) and cartilage (C2M) degradation were measured in the culture supernatants. Cultures without osteoclasts were used as background samples. Background-subtracted biomarker levels were normalized to the vehicle condition and were analyzed using analysis of variance with Tukey or Dunnett's T3 post hoc test, as applicable.

Results: Osteochondral CTX-I release was inhibited by E-64 (19% of vehicle, p = 0.0008), GM6001 (51% of vehicle, p = 0.013), and E-64/GM6001 combined (4% of vehicle, p = 0.0007)-similarly to bone CTX-I release. Diphyllin also inhibited osteochondral CTX-I release (48% of vehicle, p = 0.014), albeit less than on bone (4% of vehicle, p < 0.0001). Osteochondral C2M release was only inhibited by E-64 (49% of vehicle, p = 0.07) and GM6001 (14% of vehicle, p = 0.006), with complete abrogation when combined (0% of vehicle, p = 0.004). Cartilage C2M release was non-significantly inhibited by E-64 (69% of vehicle, p = 0.98) and was completely abrogated by GM6001 (0% of vehicle, p = 0.16).

Conclusions: Our study supports that osteoclasts can resorb non-calcified and calcified cartilage independently of acidification. We demonstrated both MMP-mediated and cysteine protease-mediated resorption of calcified cartilage. Osteoclast functionality was highly dependent on the resorbed substrate, as different ECMs required different osteoclast processes for degradation. Our novel culture system has potential to facilitate drug and biomarker development aimed at rheumatic diseases, e.g. osteoarthritis, where pathological osteoclast processes in specific joint compartments may contribute to the disease process.
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http://dx.doi.org/10.1186/s13075-018-1564-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5894194PMC
April 2018

Prospective evaluation of a combination of fungal biomarkers for the diagnosis of invasive fungal disease in high-risk haematology patients.

Mycoses 2018 Sep 26;61(9):623-632. Epub 2018 Jun 26.

Department of Infectious Diseases, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

We prospectively evaluated a combination of fungal biomarkers in adult haematology patients with focus on their clinical utility at different time points during the course of infection. In total, 135 patients were monitored once to twice weekly for serum (1-3)-ß-d-glucan (BG), galactomannan (GM), bis-methyl-gliotoxin and urinary d-arabinitol/l-arabinitol ratio. In all, 13 cases with proven or probable invasive fungal disease (IFD) were identified. The sensitivity of BG and GM at the time of diagnosis (TOD) was low, but within 2 weeks from the TOD the sensitivity of BG was 92%. BG >800 pg/mL was highly specific for IFD. At a pre-test probability of 12%, both BG and GM had negative predictive values (NPV) >0.9 but low positive predictive values (PPV). In a subgroup analysis of patients with clinically suspected IFD (pre-test probability of 35%), the NPV was lower, but the PPV for BG was 0.86 at cut-off 160 pg/mL. Among IFD patients, 91% had patterns of consecutively positive and increasing BG levels. Bis-methyl-gliotoxin was undetectable in 15 patients with proven, probable and possible IA. To conclude, BG was the superior fungal marker for IFD diagnosis. Quantification above the limit of detection and graphical evaluation of the pattern of dynamics are warranted in the interpretation of BG results.
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http://dx.doi.org/10.1111/myc.12773DOI Listing
September 2018

Agonistic targeting of TLR1/TLR2 induces p38 MAPK-dependent apoptosis and NFκB-dependent differentiation of AML cells.

Blood Adv 2017 Oct 18;1(23):2046-2057. Epub 2017 Oct 18.

Department of Clinical Genetics and.

Acute myeloid leukemia (AML) is associated with poor survival, and there is a strong need to identify disease vulnerabilities that might reveal new treatment opportunities. Here, we found that Toll-like receptor 1 (TLR1) and TLR2 are upregulated on primary AML CD34CD38 cells relative to corresponding normal bone marrow cells. Activating the TLR1/TLR2 complex by the agonist Pam3CSK4 in -driven human AML resulted in induction of apoptosis by p38 MAPK-dependent activation of Caspase 3 and myeloid differentiation in a NFκB-dependent manner. By using murine AML cells, we demonstrate that p53 is dispensable for Pam3CSK4-induced apoptosis and differentiation. Moreover, murine -driven AML cells also were forced into apoptosis and differentiation on TLR1/TLR2 activation, demonstrating that the antileukemic effects observed were not confined to -rearranged AML. We further evaluated whether Pam3CSK4 would exhibit selective antileukemic effects. Ex vivo Pam3CSK4 treatment inhibited murine and human leukemia-initiating cells, whereas murine normal hematopoietic stem and progenitor cells (HSPCs) were relatively less affected. Consistent with these findings, primary human AML cells across several genetic subtypes of AML were more vulnerable for TLR1/TLR2 activation relative to normal human HSPCs. In the AML mouse model, treatment with Pam3CSK4 provided proof of concept for in vivo therapeutic efficacy. Our results demonstrate that TLR1 and TLR2 are upregulated on primitive AML cells and that agonistic targeting of TLR1/TLR2 forces AML cells into apoptosis by p38 MAPK-dependent activation of Caspase 3, and differentiation by activating NFκB, thus revealing a new putative strategy for therapeutically targeting AML cells.
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http://dx.doi.org/10.1182/bloodadvances.2017006148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728277PMC
October 2017

CD36 defines primitive chronic myeloid leukemia cells less responsive to imatinib but vulnerable to antibody-based therapeutic targeting.

Haematologica 2018 03 28;103(3):447-455. Epub 2017 Dec 28.

Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Sweden

Tyrosine kinase inhibitors (TKIs) are highly effective for the treatment of chronic myeloid leukemia (CML), but very few patients are cured. The major drawbacks regarding TKIs are their low efficacy in eradicating the leukemic stem cells responsible for disease maintenance and relapse upon drug cessation. Herein, we performed ribonucleic acid sequencing of flow-sorted primitive (CD34CD38) and progenitor (CD34 CD38) chronic phase CML cells, and identified transcriptional upregulation of 32 cell surface molecules relative to corresponding normal bone marrow cells. Focusing on novel markers with increased expression on primitive CML cells, we confirmed upregulation of the scavenger receptor CD36 and the leptin receptor by flow cytometry. We also delineate a subpopulation of primitive CML cells expressing CD36 that is less sensitive to imatinib treatment. Using CD36 targeting antibodies, we show that the CD36 positive cells can be targeted and killed by antibody-dependent cellular cytotoxicity. In summary, CD36 defines a subpopulation of primitive CML cells with decreased imatinib sensitivity that can be effectively targeted and killed using an anti-CD36 antibody.
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http://dx.doi.org/10.3324/haematol.2017.169946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830390PMC
March 2018

[Chronic myeloid leukemia – a model disease for targeted therapy].

Lakartidningen 2017 09 21;114. Epub 2017 Sep 21.

Karolinska Universitetssjukhuset - Hematologiskt Centrum Stockholm, Sweden Karolinska Universitetssjukhuset - Hematologiskt Centrum Stockholm, Sweden.

Chronic myeloid leukemia - a model disease for targeted therapy Chronic myeloid leukemia (CML) pioneered as the first human malignancy linked to a specific cytogenetic aberration (the Philadelphia chromosome), which led the way to specific targeted therapies with imatinib (Glivec) and later tyrosine kinase inhibitors (TKI). Continuous TKI administration, blocking the oncogenic fusion protein Bcr-Abl, has revolutionized the outcome of CML, transforming an almost uniformly deadly disease into a chronic disorder with a near to normal life expectancy for many patients. There are now indications that, in a portion of patients achieving deep molecular responses, TKI treatment can be stopped without signs of relapse, indicating that these drugs may indeed induce cure. This is of particular importance since adverse events related to long-term TKI therapy, compromising quality of life, are now being increasingly recognized. With the recent introduction of generics the price of imatinib has dropped by more than 95% in Sweden, making an already cost effective treatment even more attractive.
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September 2017

Targeting NSG Mice Engrafting Cells with a Clinically Applicable Lentiviral Vector Corrects Osteoclasts in Infantile Malignant Osteopetrosis.

Hum Gene Ther 2018 08 3;29(8):938-949. Epub 2017 Oct 3.

1 Department of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund University , Lund, Sweden.

Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by nonfunctional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to develop a clinically applicable lentiviral vector expressing TCIRG1 to correct osteoclast function in IMO. Two mammalian promoters were compared: elongation factor 1α short (EFS) promoter and chimeric myeloid promoter (ChimP). EFS promoter was chosen for continued experiments, as it performed better. IMO osteoclasts corrected in vitro by a TCIRG1-expressing lentiviral vector driven by EFS (EFS-T) restored Ca release to 92% and the levels of the bone degradation product CTX-I to 95% in the media compared to control osteoclasts. IMO CD34 cells from five patients transduced with EFS-T were transplanted into NSG mice. Bone marrow was harvested 9-19 weeks after transplantation, and human CD34 cells were selected, expanded, and seeded on bone slices. Vector-corrected IMO osteoclasts had completely restored Ca release. CTX-I levels in the media were 33% compared to normal osteoclasts. Thus, in summary, evidence is provided that transduction of IMO CD34+ cells with the clinically applicable EFS-T vector leads to full rescue of osteoclasts in vitro and partial rescue of osteoclasts generated from NSG mice engrafting hematopoietic cells. This supports the continued clinical development of gene therapy for IMO.
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http://dx.doi.org/10.1089/hum.2017.053DOI Listing
August 2018

Tyrosine kinase inhibitor therapy-induced changes in humoral immunity in patients with chronic myeloid leukemia.

J Cancer Res Clin Oncol 2017 Aug 23;143(8):1543-1554. Epub 2017 Mar 23.

Hematology Research Unit Helsinki, Department of Hematology, University of Helsinki and Helsinki University Hospital Comprehensive Cancer Center, Haartmaninkatu 8, 00290, Helsinki, Finland.

Purpose: Tyrosine kinase inhibitors (TKIs) have well-characterized immunomodulatory effects on T and NK cells, but the effects on the humoral immunity are less well known. In this project, we studied TKI-induced changes in B cell-mediated immunity.

Methods: We collected peripheral blood (PB) and bone marrow (BM) samples from chronic myeloid leukemia (CML) patients before and during first-line imatinib (n = 20), dasatinib (n = 16), nilotinib (n = 8), and bosutinib (n = 12) treatment. Plasma immunoglobulin levels were measured, and different B cell populations in PB and BM were analyzed with flow cytometry.

Results: Imatinib treatment decreased plasma IgA and IgG levels, while dasatinib reduced IgM levels. At diagnosis, the proportion of patients with IgA, IgG, and IgM levels below the lower limit of normal (LLN) was 0, 11, and 6% of all CML patients, respectively, whereas at 12 months timepoint the proportions were 6% (p = 0.13), 31% (p = 0.042) and 28% (p = 0.0078). Lower initial Ig levels predisposed to the development of hypogammaglobulinemia during TKI therapy. Decreased Ig levels in imatinib-treated patients were associated with higher percentages of immature BM B cells. The patients, who had low Ig levels during the TKI therapy, had significantly more frequent minor infections during the follow-up compared with the patients with normal Ig values (33% vs. 3%, p = 0.0016). No severe infections were reported, except recurrent upper respiratory tract infections in one imatinib-treated patient, who developed severe hypogammaglobulinemia.

Conclusions: TKI treatment decreases plasma Ig levels, which should be measured in patients with recurrent infections.
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http://dx.doi.org/10.1007/s00432-017-2378-6DOI Listing
August 2017

Combination of hand-held probe and microscopy for fluorescence guided surgery in the brain tumor marginal zone.

Photodiagnosis Photodyn Ther 2017 Jun 20;18:185-192. Epub 2017 Feb 20.

Department of Biomedical Engineering, Linköping University, Sweden.

Background: Visualization of the tumor is crucial for differentiating malignant tissue from healthy brain during surgery, especially in the tumor marginal zone. The aim of the study was to introduce a fluorescence spectroscopy-based hand-held probe (HHF-probe) for tumor identification in combination with the fluorescence guided resection surgical microscope (FGR-microscope), and evaluate them in terms of diagnostic performance and practical aspects of fluorescence detection.

Material And Methods: Eighteen operations were performed on 16 patients with suspected high-grade glioma. The HHF-probe and the FGR-microscope were used for detection of protoporphyrin (PpIX) fluorescence induced by 5-aminolevulinic acid (5-ALA) and evaluated against histopathological analysis and visual grading done through the FGR-microscope by the surgeon. A ratio of PpIX fluorescence intensity to the autofluorescence intensity (fluorescence ratio) was used to quantify the spectra detected by the probe.

Results: Fluorescence ratio medians (range 0 - 40) measured by the probe were related to the intensity of the fluorescence in the FGR-microscope, categorized as "none" (0.3, n=131), "weak" (1.6, n=34) and "strong" (5.4, n=28). Of 131 "none" points in the FGR-microscope, 88 (67%) exhibited fluorescence with the HHF-probe. For the tumor marginal zone, the area under the receiver operator characteristics (ROC) curve was 0.49 for the FGR-microscope and 0.65 for the HHF-probe.

Conclusions: The probe was integrated in the established routine of tumor resection using the FGR-microscope. The HHF-probe was superior to the FGR-microscope in sensitivity; it detected tumor remnants after debulking under the FGR-microscope. The combination of the HHF-probe and the FGR-microscope was beneficial especially in the tumor marginal zone.
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http://dx.doi.org/10.1016/j.pdpdt.2017.01.188DOI Listing
June 2017

Forced expression of human macrophage colony-stimulating factor in CD34 cells promotes monocyte differentiation in vitro and in vivo but blunts osteoclastogenesis in vitro.

Eur J Haematol 2017 May 6;98(5):517-526. Epub 2017 Mar 6.

Department of Molecular Medicine and Gene Therapy, BMC A12, Lund University, Lund, Sweden.

Objectives: Here, we tested the hypothesis that human M-CSF (hM-CSF) overexpressed in cord blood (CB) CD34 cells would induce differentiation and survival of monocytes and osteoclasts in vitro and in vivo.

Methods: Human M-CSF was overexpressed in cord blood CD34 cells using a lentiviral vector.

Results: We show that LV-hM-CSF-transduced CB CD34 cells expand 3.6- and 8.5-fold more with one or two exposures to the hM-CSF-expressing vector, respectively, when compared to control cells. Likewise, LV-hM-CSF-transduced CB CD34 cells show significantly higher levels of monocytes. In addition, these cells produced high levels of hM-CSF. Furthermore, they are able to differentiate into functional bone-resorbing osteoclasts in vitro. However, osteoclast differentiation and bone resorption were blunted compared to control CD34 cells receiving exogenous hM-CSF. NSG mice engrafted with LV-hM-CSF-transduced CB CD34 cells have physiological levels of hM-CSF production that result in an increase in the percentage of human monocytes in peripheral blood and bone marrow as well as in the spleen, lung and liver.

Conclusion: In summary, ectopic production of human M-CSF in CD34 cells promotes cellular expansion and monocyte differentiation in vitro and in vivo and allows for the formation of functional osteoclasts, albeit at reduced levels, without an exogenous source of M-CSF, in vitro.
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http://dx.doi.org/10.1111/ejh.12867DOI Listing
May 2017

Early BCR-ABL1 Transcript Decline after 1 Month of Tyrosine Kinase Inhibitor Therapy as an Indicator for Treatment Response in Chronic Myeloid Leukemia.

PLoS One 2017 30;12(1):e0171041. Epub 2017 Jan 30.

Hematology Research Unit Helsinki, Department of Hematology, University of Helsinki and Helsinki University Central Hospital Comprehensive Cancer Center, Helsinki, Finland.

In chronic myeloid leukemia (CML), early treatment prediction is important to identify patients with inferior overall outcomes. We examined the feasibility of using reductions in BCR-ABL1 transcript levels after 1 month of tyrosine kinase inhibitor (TKI) treatment to predict therapy response. Fifty-two first-line TKI-treated CML patients were included (imatinib n = 26, dasatinib n = 21, nilotinib n = 5), and BCR-ABL1 transcript levels were measured at diagnosis (dg) and 1, 3, 6, 12, 18, 24, and 36 months. The fold change of the BCR-ABL1 transcripts at 1 month compared to initial BCR-ABL1 transcript levels was used to indicate early therapy response. In our cohort, 21% of patients had no decrease in BCR-ABL1 transcript levels after 1 month and were classified as poor responders. Surprisingly, these patients had lower BCR-ABL1 transcript levels at dg compared to responders (31% vs. 48%, p = 0.0083). Poor responders also significantly more often had enlarged spleen (55% vs. 15%; p<0.01) and a higher percentage of Ph+ CD34+CD38- cells in the bone marrow (91% vs. 75%, p<0.05). The major molecular response rates were inferior in the poor responders (at 12m 18% vs. 64%, p<0.01; 18m 27% vs. 75%, p<0.01; 24m 55% vs. 87%, p<0.01). In conclusion, early treatment response analysis defines a biologically distinct patient subgroup with inferior long-term outcomes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171041PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5279791PMC
August 2017

Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML.

Blood 2017 04 25;129(17):2384-2394. Epub 2017 Jan 25.

Division of Molecular Hematology, and.

Understanding leukemia heterogeneity is critical for the development of curative treatments as the failure to eliminate therapy-persistent leukemic stem cells (LSCs) may result in disease relapse. Here we have combined high-throughput immunophenotypic screens with large-scale single-cell gene expression analysis to define the heterogeneity within the LSC population in chronic phase chronic myeloid leukemia (CML) patients at diagnosis and following conventional tyrosine kinase inhibitor (TKI) treatment. Our results reveal substantial heterogeneity within the putative LSC population in CML at diagnosis and demonstrate differences in response to subsequent TKI treatment between distinct subpopulations. Importantly, LSC subpopulations with myeloid and proliferative molecular signatures are proportionally reduced at a higher extent in response to TKI therapy compared with subfractions displaying primitive and quiescent signatures. Additionally, cell surface expression of the CML stem cell markers CD25, CD26, and IL1RAP is high in all subpopulations at diagnosis but downregulated and unevenly distributed across subpopulations in response to TKI treatment. The most TKI-insensitive cells of the LSC compartment can be captured within the CD45RA fraction and further defined as positive for CD26 in combination with an aberrant lack of cKIT expression. Together, our results expose a considerable heterogeneity of the CML stem cell population and propose a LinCD34CD38CD45RAcKITCD26 population as a potential therapeutic target for improved therapy response.
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http://dx.doi.org/10.1182/blood-2016-07-728873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484462PMC
April 2017

The impact of socio-economic factors on treatment choice and mortality in chronic myeloid leukaemia.

Eur J Haematol 2017 Apr 16;98(4):398-406. Epub 2017 Jan 16.

Unit of Haematology, Department of Medical Sciences, Uppsala University, Uppsala, Sweden.

Purpose: To evaluate the influence of socio-economic variables on treatment selection and survival of patients with chronic myeloid leukaemia (CML).

Methods: Using information available in population-based Swedish registries, we evaluated indices of health, education and economy from the 980 patients in the Swedish CML register diagnosed between 2002 and 2012. Apart from internal comparisons, five age-, gender- and region-matched control subjects per patient served as control cohort. Median follow-up time from CML diagnosis was 4.8 years.

Results: Among patients with CML, low personal or household income, short education, living alone, poor performance status and high age (>60 years) were significantly associated with an inferior survival (in univariate analyses). However, similar findings were noted also in the matched control group, and in comparisons adjusted for calendar year, age and performance status, socio-economic variables were not significantly associated with CML survival. Meanwhile, both education and income were independently linked to TKI treatment overall and to upfront treatment with second-generation TKIs.

Conclusions: In conclusion, socio-economic conditions were associated with survival in the studied CML cohort but these associations could be explained by differences at baseline. Meanwhile, socio-economic conditions appeared to influence treatment choice.
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http://dx.doi.org/10.1111/ejh.12845DOI Listing
April 2017

Plasma proteomics in CML patients before and after initiation of tyrosine kinase inhibitor therapy reveals induced Th1 immunity and loss of angiogenic stimuli.

Leuk Res 2016 11 27;50:95-103. Epub 2016 Sep 27.

Dept. of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Background And Aims: The simultaneous measurement of many proteins is now possible using multiplex assays. In this pilot study we investigated a total of 124 proteins in plasma from chronic myeloid leukemia (CML) patients with the purpose of identifying proteins that are differently expressed at diagnosis and after tyrosine kinase inhibitor (TKI) treatment initiation.

Methods: Samples were taken from 14 CML patients at diagnosis and after three months of TKI treatment (imatinib or dasatinib). Samples were analyzed by Mesoscale Discovery, Myriad RBM MAP technology and Olink Proseek.

Results: Multiple plasma proteins were differentially expressed before and after initiation of TKI therapy. Protein patterns demonstrated a possible shift towards Th1-immunity and reduced angiogenic stimuli. Further, some plasma proteins were identified that can be of potential interest to study further for biologic, prognostic or therapeutic significance such as E-selectin, uPAR, growth hormone and carbonic anhydrase IX.

Conclusions: Plasma proteomics seems feasible and useful in CML patients, both for studying patterns of protein expression and for identifying single proteins differentially expressed before and after treatment. Plasma proteomics may be useful to map disease activity and biological processes. Hence, plasma proteomics can be used to understand drug mechanisms and treatment responses in CML.
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http://dx.doi.org/10.1016/j.leukres.2016.09.019DOI Listing
November 2016

IL1RAP antibodies block IL-1-induced expansion of candidate CML stem cells and mediate cell killing in xenograft models.

Blood 2016 12 12;128(23):2683-2693. Epub 2016 Sep 12.

Department of Clinical Genetics, Lund University, Lund, Sweden.

Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors, but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Interleukin-1 receptor accessory protein (IL1RAP; IL1R3) is a coreceptor of interleukin-1 receptor type 1 and has been found upregulated on CML stem cells. Here, we show that primitive (CD34CD38) CML cells, in contrast to corresponding normal cells, express a functional interleukin-1 (IL-1) receptor complex and respond with NF-κB activation and marked proliferation in response to IL-1. IL1RAP antibodies that inhibit IL-1 signaling could block these effects. In vivo administration of IL1RAP antibodies in mice transplanted with chronic and blast phase CML cells resulted in therapeutic effects mediated by murine effector cells. These results provide novel insights into the role of IL1RAP in CML and a strong rationale for the development of an IL1RAP antibody therapy to target residual CML stem cells.
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http://dx.doi.org/10.1182/blood-2015-11-679985DOI Listing
December 2016

Regulation and Function of Lentiviral Vector-Mediated TCIRG1 Expression in Osteoclasts from Patients with Infantile Malignant Osteopetrosis: Implications for Gene Therapy.

Calcif Tissue Int 2016 12 19;99(6):638-648. Epub 2016 Aug 19.

Nordic Bioscience, Herlev, Denmark.

Infantile malignant osteopetrosis (IMO) is a rare, recessive disorder characterized by increased bone mass caused by dysfunctional osteoclasts. The disease is most often caused by mutations in the TCIRG1 gene encoding a subunit of the V-ATPase involved in the osteoclasts capacity to resorb bone. We previously showed that osteoclast function can be restored by lentiviral vector-mediated expression of TCIRG1, but the exact threshold for restoration of resorption as well as the cellular response to vector-mediated TCIRG1 expression is unknown. Here we show that expression of TCIRG1 protein from a bicistronic TCIRG1/GFP lentiviral vector was only observed in mature osteoclasts, and not in their precursors or macrophages, in contrast to GFP expression, which was observed under all conditions. Thus, vector-mediated TCIRG1 expression appears to be post-transcriptionally regulated, preventing overexpression and/or ectopic expression and ensuring protein expression similar to that of wild-type osteoclasts. Codon optimization of TCIRG1 led to increased expression of mRNA but lower levels of protein and functional rescue. When assessing the functional rescue threshold in vitro, addition of 30 % CB CD34 cells to IMO CD34 patient cells was sufficient to completely normalize resorptive function after osteoclast differentiation. From both an efficacy and a safety perspective, these findings will clearly be of benefit during further development of gene therapy for osteopetrosis.
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http://dx.doi.org/10.1007/s00223-016-0187-6DOI Listing
December 2016
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