Summary Cancer stem cells, also known as leukemic stem cells (LSC) in the context of leukemias, are an emerging topic in translational oncology and hematology. The Ludwig Boltzmann Institute for Hematology and Oncology (LBI HO) was established in 2008 with the aim to translate LSC concepts into clinical practice. Major specific aims of the LBI HO are to identify LSC in various blood cell disorders and to improve anti-leukemic therapies by establishing LSC-targeting and LSC-eradicating approaches with the ultimate aim to translate these concepts into clinical practice. In addition, the LBI HO identified a number of diagnostic and prognostic LSC markers in various blood cell malignancies. Members of the LBI HO have also developed precision medicine tools and personalized medicine approaches around LSC in applied hematology. As a result, diagnosis, prognostication and therapy have improved in the past 10 years. Major disease models are myeloid leukemias and mast cell neoplasms. Finally, the LBI HO consortium launched several projects in the field of open innovation in science where patient-derived initiatives and their input supported the scientific community. Key aims for the future of the LBI HO are to develop LSC-related concepts and strategies further, with the long-term vision to cure more patients with hematologic malignancies.

In orthopedic surgery, implant infections are a serious issue and difficult to treat. The aim of this study was to use superparamagnetic nanoporous silica nanoparticles (MNPSNP) as candidates for directed drug delivery. Currently, short blood circulation half-life due to interactions with the host's immune system hinder nanoparticles in general from being clinically used. PEGylation is an approach to reduce these interactions and to enhance blood circulation time. The effect of PEGylation of the used 68Ga-labelled MNPSNP on the distribution and implant accumulation was examined by PET/CT imaging and gamma counting in an implant mouse model.Female Balb/c mice (n=24) received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. On day one, 12 of these mice received an additional clodronate®-injection for macrophage depletion. On the second postoperative day, mice were anaesthetized and MNPSNP (native or PEGylated) injected intravenously, followed by a dynamic PET-scan over 60 minutes, a CT- and a static PET-scan at 120 min. As control, 12 mice received only 68Ga-MNPSNP (native or PEGylated). Gamma counting of inner organs, urine, blood and implant area was performed as further final analysis. Although PEGylation of the nanoparticles already resulted in lower liver uptakes, both variants of 68Ga-labeled MNPSNP accumulated in liver and spleen. Combination of PEGylation with clodronate®-injection led to a highly significant effect whereas clodronate®-injection alone could not reveal significant differences. In gamma counting, a significantly higher %I.D./g was found for the tissue surrounding the magnetic implants compared to the titanium control, although in a low range. PEGylation and/or clodronate®-injection revealed no significant differences regarding nanoparticle accumulation at the implantation site. PEGylation increases circulation time, but MNPSNP accumulation at the implant site was still insufficient for treatment of infections. Additional efforts have to further increase circulation time and local accumulation.Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 280642759).

PDF file - 162K, Supplementary Figure S1A: Expression of CD52 on CD34+/CD38+ cells in MDS and AML Supplementary Figure S1B: Expression of CD52 on CD34+/CD38+ cells in MDS, AML and control samples.

PDF file - 61K, Supplementary Table S9: Detection of del(5q) in sorted cells of MDS patients by FISH.

PDF file - 143K, Supplementary Figure S2A: Effects of alemtuzumab on growth of HL60 cells transfected with empty vector or NRAS Q61K Supplementary Figure S2B: Effects of alemtuzumab in combination with IgG on growth of MDS and AML stem cells Supplementary Figure S2C: Correlation between CD52 surface expression and the cytotoxic effect of alemtuzumab.

PDF - 94K, Supplementary Methods.

PDF file - 103K, Supplementary Figure S3: Expression of CD52 on CD34+/CD38− cells in different WHO subtypes of MDS and AML.

PDF file - 83K, Supplementary Table S6: Expression of CD52, CD123 and CLL-1 on CD34+/CD38−/CD90+/CD45RA− cells in patients with MDS and on CD34+/CD38−/CD90−/CD45RA− cells in patients with AML.

Myeloproliferative neoplasms (MPN) are characterized by uncontrolled expansion of myeloid cells, disease-related mutations in certain driver-genes including JAK2, CALR, and MPL, and a substantial risk to progress to secondary acute myeloid leukemia (sAML). Although behaving as stem cell neoplasms, little is known about disease-initiating stem cells in MPN. We established the phenotype of putative CD34+ /CD38- stem cells and CD34+ /CD38+ progenitor cells in MPN. A total of 111 patients with MPN suffering from polycythemia vera, essential thrombocythemia, or primary myelofibrosis (PMF) were examined. In almost all patients tested, CD34+ /CD38- stem cells expressed CD33, CD44, CD47, CD52, CD97, CD99, CD105, CD117, CD123, CD133, CD184, CD243, and CD274 (PD-L1). In patients with PMF, MPN stem cells often expressed CD25 and sometimes also CD26 in an aberrant manner. MPN stem cells did not exhibit substantial amounts of CD90, CD273 (PD-L2), CD279 (PD-1), CD366 (TIM-3), CD371 (CLL-1), or IL-1RAP. The phenotype of CD34+ /CD38- stem cells did not change profoundly during progression to sAML. The disease-initiating capacity of putative MPN stem cells was confirmed in NSGS mice. Whereas CD34+ /CD38- MPN cells engrafted in NSGS mice, no substantial engraftment was produced by CD34+ /CD38+ or CD34- cells. The JAK2-targeting drug fedratinib and the BRD4 degrader dBET6 induced apoptosis and suppressed proliferation in MPN stem cells. Together, MPN stem cells display a unique phenotype, including cytokine receptors, immune checkpoint molecules, and other clinically relevant target antigens. Phenotypic characterization of neoplastic stem cells in MPN and sAML should facilitate their enrichment and the development of stem cell-eradicating (curative) therapies.

PDF file - 88K, Supplementary Table S7: Expression of the surface antigen CD52 on CD34+/CD38− cells and CD34+/CD38+ cells in CML, CMML, MDS/MPN-U, MPN, AUL, and ALL Supplementary Table S8: Expression of CD52 on CD34+/CD38− cells, CD34+/CD38+ cells, monocytes, and blood basophils in patients with ICUS, normal/reactive bone marrow, and complete long-term remission after AML.

IntroductionThe Vienna Cancer Stem Cell Club (VCSCC) was launched by a group of scientists in Vienna in 2002.Areas coveredMajor aims of the VCSCC are to support research on cancer stem cells (CSC) in hematopoietic malignancies and to translate CSC-related markers and targets into clinical application. A primary focus of research in the VCSCC is the leukemic stem cell (LSC). Between 2013 and 2021, members of the VCSCC established a special research program on myeloproliferative neoplasms and since 2008, members of the VCSCC run the Ludwig Boltzmann Institute for Hematology and Oncology. In all these years, the VCSCC provided a robust intellectual platform for translational hematology and LSC research in Vienna. Furthermore, the VCSCC interacts with several national and international study groups and societies in the field. Representatives of the VCSCC also organized a number of international meetings and conferences on neoplastic stem cells, including LSC, in the past 15 years, and contributed to the definition and classification of CSC/LSC and related pre-malignant and malignant conditions.Expert opinionThe VCSCC will continue to advance the field and to develop LSC-detecting and LSC-eradicating concepts through which diagnosis, prognostication, and therapy of blood cancer patients should improve.

PDF file - 85K, Supplementary Table S1: Characteristics of patients with MDS Supplementary Table S2: Characteristics of patients with AML Supplementary Table S3: Characteristics of patients with CML, CMML, MPS, AUL, and ALL Supplementary Table S4: Characteristics of patients with ICUS, normal/reactive bone marrow and long-term complete remission.

Systemic mastocytosis (SM) is a heterogeneous group of disorders characterized by abnormal growth and accumulation of mast cells (MC) in various organ-systems [1][2][3].The classification of the World Health Organization (WHO) divides SM into non-advanced forms and advanced forms of the disease.Whereas patients with non-advanced SM, such as indolent SM (ISM) or smoldering SM (SSM) have an excellent prognosis, the prognosis and survival of patients with advanced SM, including aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN) and MC leukemia (MCL), are dismal [1][2][3].Despite the prognostic value of the WHO classification, availability of additional prognostic variables, and recently developed prognostic scores, it is often difficult to predict the course and prognosis in individual patients [4,5].For example, even patients with ISM who have no or only a few risk factors concerning progression, may sometimes progress to ASM or MCL.One strategy to define the expansion and progression of neoplastic cells may be to measure the dynamics of MC-related parameters such as the tryptase level or MC infiltration grade in the bone marrow (BM) [6,7].However, serum tryptase levels may also increase in ISM or SSM without signs of progression and serial BM investigations are usually not performed.It is also difficult to measure the proliferative capacity of neoplastic progenitors in patients with SM [8].Another approach may be to measure the numbers and disease-propagating ability of neoplastic stem cells in patients with SM.However, the lack of suitable in vivo models reflecting non-advanced SM and advanced SM has limited previous attempts to correlate in vivo expansion of SM cells with clinical endpoints and prognosis.The concept of leukemic stem cells (LSC) is well established in acute and chronic leukemias [9,10].More recently, we have identified LSC in patients with advanced SM, including MCL [11].In most patients with MCL, LSC were found to produce detectable engraftment in NSG mice exhibiting membrane-bound human stem cell factor (NSG SCF ) [11].We also found that engraftment

PDF file - 48K, Supplementary Table S5: The oligonucleotide primer sequences for quantitative PCR (qPCR).

PDF - 210K, Can225IgG heavy and light chain amino acid sequences.

PDF - 446K, Figure S1. Purification of can225IgG from cell culture supernatant is efficient with Protein G, but not with Protein A. Figure S2. Elution of can225IgG from Protein G columns at a low pH does not affect integrity of the antibody. Figure S3. Binding of can225IgG to the cell lines CF33, CF41, TLM-1 and BT474 in FACS. Figure S4. Can225IgG shows a slightly lower binding affinity towards canine EGFR, compared with human EGFR. Figure S5. Staining of EGFR+ cell line and canine mammary carcinoma section by can225IgG.

In most patients with chronic myeloid leukemia (CML) clonal cells can be kept under control by BCR::ABL1 tyrosine kinase inhibitors (TKI). However, overt resistance or intolerance against these TKI may occur. We identified the epigenetic reader BRD4 and its downstream-effector MYC as growth regulators and therapeutic targets in CML cells. BRD4 and MYC were found to be expressed in primary CML cells, CD34+ /CD38- leukemic stem cells (LSC), and in the CML cell lines KU812, K562, KCL22, and KCL22T315I . The BRD4-targeting drug JQ1 was found to suppress proliferation in KU812 cells and primary leukemic cells in the majority of patients with chronic phase CML. In the blast phase of CML, JQ1 was less effective. However, the BRD4 degrader dBET6 was found to block proliferation and/or survival of primary CML cells in all patients tested, including blast phase CML and CML cells exhibiting the T315I variant of BCR::ABL1. Moreover, dBET6 was found to block MYC expression and to synergize with BCR::ABL1 TKI in inhibiting the proliferation in the JQ1-resistant cell line K562. Furthermore, BRD4 degradation was found to overcome osteoblast-induced TKI resistance of CML LSC in a co-culture system and to block interferon-gamma-induced upregulation of the checkpoint antigen PD-L1 in LSC. Finally, dBET6 was found to suppress the in vitro survival of CML LSC and their engraftment in NSG mice. Together, targeting of BRD4 and MYC through BET degradation sensitizes CML cells against BCR::ABL1 TKI and is a potent approach to overcome multiple forms of drug resistance in CML LSC.

Mast cell neoplasms are one of the most frequently diagnosed malignancies in dogs. The clinical picture, course, and prognosis vary substantially among patients, depending on the anatomic site, grade and stage of the disease. The most frequently involved organ is the skin, followed by hematopoietic organs (lymph nodes, spleen, liver, and bone marrow) and mucosal sites of the oral cavity and the gastrointestinal tract. In cutaneous mast cell tumors, several grading and staging systems have been introduced. However, no comprehensive classification and no widely accepted diagnostic criteria have been proposed to date. To address these open issues and points we organized a Working Conference on canine mast cell neoplasms in Vienna in 2019. The outcomes of this meeting are summarized in this article. The proposed classification includes cutaneous mast cell tumors and their sub-variants defined by grading- and staging results, mucosal mast cell tumors, extracutaneous/extramucosal mast cell tumors without skin involvement, and mast cell leukemia (MCL). For each of these entities, diagnostic criteria are proposed. Moreover, we have refined grading and staging criteria for mast cell neoplasms in dogs based on consensus discussion. The criteria and classification proposed in this article should greatly facilitate diagnostic evaluation and prognostication in dogs with mast cell neoplasms and should thereby support management of these patients in daily practice and the conduct of clinical trials.

AbstractCanine mastocytomas (MCTs) are characterized by rapid proliferation of neoplastic mast cells (MCs) and clinical signs caused by MC‐derived mediators. In dogs suffering from MCT, histamine receptor 1 (HR1) antagonists are frequently used to control mediator‐related clinical symptoms. Previous studies have shown that the HR1 antagonists loratadine and terfenadine exert some growth‐inhibitory effects on neoplastic MCs. We examined whether other HR1 antagonists used in clinical practice (desloratadine, rupatadine, cyproheptadine, dimetindene, diphenhydramine) affect proliferation and survival of neoplastic MCs. Furthermore, we analysed whether these HR1 antagonists counteract IgE‐dependent histamine release from a MC line harbouring a functional IgE‐receptor. HR1 antagonists were applied on two canine MC lines, C2 and NI‐1, and on primary MCs obtained from three MCT samples. The HR1 antagonists desloratadine, rupatadine and cyproheptadine were found to be more potent in decreasing proliferation of C2 and NI‐1 cells when compared with dimetindene and diphenhydramine. Similar effects were seen in primary neoplastic MCs, except for diphenhydramine, which exerted more potent growth‐inhibitory effects than the other HR1 antagonists. Drug‐induced growth‐inhibition in C2 and NI‐1 cells was accompanied by apoptosis. Loratadine, desloratadine and rupatadine also suppressed IgE‐dependent histamine release in NI‐1 cells. However, drug concentrations required to elicit substantial effects on growth or histamine release were relatively high (>10 µM). Therefore, it remains unknown whether these drugs or similar, more potent, HR1‐targeting drugs can suppress growth or activation of canine neoplastic MCs in vivo.

Localized histiocytic sarcoma may occur as a primary lesion in periarticular tissues of large appendicular joints. Treatment options for the primary lesion include radical surgical excision, radiation therapy (RT), or both, in combination with chemotherapy for potential systemic metastases. In an effort to better characterize the time to progression (TTP) following surgical vs non-surgical approaches for periarticular histiocytic sarcoma (PAHS), a contemporary European population of affected dogs was retrospectively surveyed. Medical records were queried for newly-diagnosed PAHS cases undergoing surgery (predominantly limb amputation) or RT followed by systemic chemotherapy. Of 49 dogs, 34 underwent RT and 15 underwent surgery. All dogs received adjuvant chemotherapy. There was no statistically significant difference in TTP or overall survival between groups. The median TTP was 336 days for the operated dogs and 217 days for the irradiated dogs (P = .117). The median overall survival time was 398 days for the operated dogs and 240 days for the irradiated dogs (P = .142). On multi-variable analysis, the variables significantly associated with an increased risk of both tumour progression and tumour-related death were regional lymph node and distant metastasis at admission. Survival and local control rates following RT may be comparable to radical resection. These data may better inform shared decision-making processes between multi-disciplinary care providers and owners.