The development of Philadelphia chromosome-negative classical myeloproliferative neoplasms (MPN) involves an inflammatory process that facilitates outgrowth of the malignant clone and correlates with clinical outcome measures. This raises the question to which extent inflammatory circuits in MPN depend on activation of innate immune sensors. Here, we investigate whether NLRP3, which precipitates inflammasome assembly upon detection of cellular stress, drives murine JAK2V617F mutant MPN. Deletion of Nlrp3 within the hematopoietic compartment completely prevents increased IL-1β and IL-18 release in MPN. NLRP3 in JAK2V617F hematopoietic cells, but not in JAK2 wild type radioresistant cells, promotes excessive platelet production via stimulation of the direct thrombopoiesis differentiation pathway, as well as granulocytosis. It also promotes expansion of the hematopoietic stem and progenitor cell compartment despite inducing pyroptosis at the same time. Importantly, NLRP3 inflammasome activation enhances bone marrow fibrosis and splenomegaly. Pharmacological blockade of NLRP3 in fully established disease leads to regression of thrombocytosis, splenomegaly and bone marrow fibrosis. These findings suggest that NLRP3 is critical for MPN development and its inhibition represents a new therapeutic intervention for MPN patients.

Bone marrow fibrosis in myeloproliferative neoplasm (MPN), myelodysplastic syndromes (MDS), MPN/MDS overlap syndromes and acute myeloid leukemia (AML) is associated with poor prognosis and early treatment failure. Myelofibrosis (MF) is accompanied by reprogramming of multipotent bone marrow mesenchymal stromal cells (MSC) into osteoid and fiber-producing stromal cells. We demonstrate NRP2 and osteolineage marker NCAM1 (neural cell adhesion molecule 1) expression within the endosteal niche in normal bone marrow and aberrantly in MPN, MDS MPN/MDS overlap syndromes and AML (n = 99), as assessed by immunohistochemistry. Increased and diffuse expression in mesenchymal stromal cells and osteoblasts correlates with high MF grade in MPN (p < 0.05 for NRP2 and NCAM1). Single cell RNA sequencing (scRNAseq) re-analysis demonstrated NRP2 expression in endothelial cells and partial co-expression of NRP2 and NCAM1 in normal MSC and osteoblasts. Potential ligands included transforming growth factor β1 (TGFB1) from osteoblasts and megakaryocytes. Murine ThPO and JAK2V617F myelofibrosis models showed co-expression of Nrp2 and Ncam1 in osteolineage cells, while fibrosis-promoting MSC only express Nrp2. In vitro experiments with MC3T3-E1 pre-osteoblasts and analysis of Nrp2−/− mouse femurs suggest that Nrp2 is functionally involved in osteogenesis. In summary, NRP2 represents a potential novel druggable target in patients with myelofibrosis.

PDF file - 2976K, HCC cell lines derived from c-myc OVA tg+ mice express OVA and functional antigen presenting molecules.

PDF file - 4662K, Three week old mice present established multifocal hepatocellular carcinomas and neonatal mice express OVA mRNA in their livers.

PDF file - 1818K, Activated T cells and tumor infiltrating lymphocytes express PD-1, B7-H1 (PD-L1), CD137 and OX40.

PDF file - 1620K, Combo3 treatment induces infiltrates of blastic T lymphocytes and increases the CD8/Treg ratio.

Nestin is an intermediate filament protein, which was originally detected in neuroepithelial stem cells. Besides its use as a phenotypic marker of mesenchymal stem cells in the hematopoeitic stem cell niche, the functional interpretation of nestin+ cells remains elusive. We investigated the cellular expression of nestin in bone marrow trephine biopsies of MPN patients, following myeloablation at a stage of hypocellularity during early regeneration. Here, nestin is highly expressed in mature osteocytes, arteriolar endothelial and perivascular cells and small capillaries within the bone marrow space, but not in sinusoid lining cells. This is in stark contrast to nestin expression pattern in myeloproliferative neoplasms that show hypercellularity due to oncogenic driver mutations. Here, nestin is expressed exclusively in endothelial cells of arterioles, but not in osteocytes or small capillaries. Thus, the pattern of nestin expression following myeloablation inversely correlates with cellularity in the bone marrow. This nestin expression pattern is mimicking early postnatal transcriptional programming during bone marrow development. We show that nestin expression in osteocytes occurs across different species following transplant and also in bone marrow metastasis.

Recently, immunotherapeutic approaches have become a feasible option for a subset of pediatric cancer patients. Low MHC class I expression hampers the use of immunotherapies relying on antigen presentation. A well-established stemness score (mRNAsi) was determined using the bulk transcriptomes of 1134 pediatric small round blue cell tumors. Interestingly, MHC class I gene expression (HLA-A/-B/-C) was correlated negatively with mRNAsi throughout all diagnostic entities: neuroblastomas (NB) (n = 88, r = −0.41, p < 0.001), the Ewing’s sarcoma family of tumors (ESFT) (n = 117, r = −0.46, p < 0.001), rhabdomyosarcomas (RMS) (n = 158, r = −0.5, p < 0.001), Wilms tumors (WT) (n = 224, r = −0.39, p < 0.001), and central nervous system-primitive neuroectodermal tumors CNS-PNET (r = −0.49, p < 0.001), with the exception of medulloblastoma (MB) (n = 76, r = −0.24, p = 0.06). The negative correlation of MHC class I and mRNAsi was independent of clinical features in NB, RMS, and WT. In NB and WT, increased MHC class I was correlated negatively with tumor stage. RMS patients with a high expression of MHC class I and abundant CD8 T cells showed a prolonged overall survival (n = 148, p = 0.004). Possibly, low MHC class I expression and stemness in pediatric tumors are remnants of prenatal tumorigenesis from multipotent precursor cells. Further studies are needed to assess the usefulness of stemness and MHC class I as predictive markers.

Blastic plasmacytoid dendritic-cell neoplasm (BPDCN) is an extremely rare disease that originates from dendritic cells and is associated with a poor overall survival (OS). Diagnostic and therapeutic standards are less well-established in comparison to other leukemic conditions and standards of care are lacking. Morphologic and molecular similarities to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) are hard to distinguish. We here report a BPDCN patient with a long, challenging diagnostic period. While bone marrow biopsies initially failed to prove the correct diagnosis, a cutaneous biopsy finally identified a CD45+/CD56+/CD4+/CD123+/CD33+/MPO- population suggestive of BPDCN which was confirmed by flow cytometry. Molecular analysis revealed an ASXL-1, TET2 and SRSF2-mutation, cytogenetic analysis showed a normal karyotype. Treatment with the recently approved CD123-cytotoxin Tagraxofusp showed initially a very good response. This case reflects diagnostic and therapeutic difficulties in BPDCN as very rare, easily misdiagnosed neoplasia and the need for precise diagnostic care.

Overall survival of gastric cancer remains low, as patients are often diagnosed with advanced stage disease. In this review, we give an overview of current research on biomarkers in gastric cancer and their implementation in treatment strategies. The HER2-targeting trastuzumab is the first molecular targeted agent approved for gastric cancer treatment. Other promising biomarkers for targeted therapies that have shown relevance in clinical trials are VEGF and Claudin 18.2. Expression of MET has been shown to be a negative prognostic factor in gastric cancer. Targeting the PD-1/PD-L1 pathway with immune checkpoint inhibitors has proven efficacy in advanced gastric cancer. Recent technology advances allow the detection of circulating tumor cells that may be used as diagnostic and prognostic indicators and for therapy monitoring in gastric cancer patients. Prognostic molecular subtypes of gastric cancer have been identified using genomic data. In addition, transcriptome profiling has allowed a comprehensive characterization of the immune and stromal microenvironment in gastric cancer and development of novel risk scores. These prognostic and predictive markers highlight the rapidly evolving field of research in gastric cancer, promising improved treatment stratification and identification of molecular targets for individualized treatment in gastric cancer.

Pediatric tumors frequently arise from embryonal cells, often displaying a stem cell-like (“small round blue”) morphology in tissue sections. Because recently “stemness” has been associated with a poor immune response in tumors, we investigated the association of prognostic gene expression, stemness and the immune microenvironment systematically using transcriptomes of 4068 tumors occurring mostly at the pediatric and young adult age. While the prognostic landscape of gene expression (PRECOG) and infiltrating immune cell types (CIBERSORT) is similar to that of tumor entities occurring mainly in adults, the patterns are distinct for each diagnostic entity. A high stemness score (mRNAsi) correlates with clinical and morphologic subtype in Wilms tumors, neuroblastomas, synovial sarcomas, atypical teratoid rhabdoid tumors and germ cell tumors. In neuroblastomas, a high mRNAsi is associated with shortened overall survival. In Wilms tumors a high mRNAsi correlates with blastemal morphology, whereas tumors with predominant epithelial or stromal differentiation have a low mRNAsi and a high percentage of M2 type macrophages. This could be validated in Wilms tumor tissue (n = 78). Here, blastemal areas are low in M2 macrophage infiltrates, while nearby stromal differentiated areas contain abundant M2 macrophages, suggesting local microanatomic regulation of the immune response.

Bone marrow (BM) fibrosis in myeloproliferative neoplasms (MPNs) is associated with a poor prognosis. The development of myelofibrosis and differentiation of mesenchymal stromal cells to profibrotic myofibroblasts depends on macrophages. Here, we compared macrophage frequencies in BM biopsies of MPN patients and controls (patients with non-neoplastic processes), including primary myelofibrosis (PMF, n = 18), essential thrombocythemia (ET, n = 14), polycythemia vera (PV, n = 12), and Philadelphia chromosome–positive chronic myeloid leukemia (CML, n = 9). In PMF, CD68-positive macrophages were greatly increased compared to CML (p = 0.017) and control BM (p < 0.001). Similar findings were observed by CD163 staining (PMF vs. CML: p = 0.017; PMF vs. control: p < 0.001). Moreover, CD68-positive macrophages were increased in PV compared with ET (p = 0.009) and reactive cases (p < 0.001). PMF had higher frequencies of macrophages than PV (CD68: p < 0.001; CD163: p < 0.001) and ET (CD68: p < 0.001; CD163: p < 0.001). CD163 and CD68 were often co-expressed in macrophages with stellate morphology in Philadelphia chromosome–negative MPN, resulting in a sponge-like reticular network that may be a key regulator of unbalanced hematopoiesis in the BM space and may explain differences in cellularity and clinical course.

This year's symposium of the working group anemia of the German Geriatric Society (DGG) aimed to underline the multicausality of anemia in the aged and to highlight definition parallels with geriatric syndromes. For these reasons, nutritional and malignant causes for anemia were discussed and the influence of oxidative stress on the development of anemia was underlined. The need for ongoing research in the field of anemia in the aged was emphasized by the lack of perioperative transfusion strategies in geriatric patients.

Functional contributions of individual members of the bone marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPN) are incompletely understood. Consequently, we aimed to generate a comprehensive map of the stroma in MPN/MF on a single cell level. Our analysis revealed two distinct mesenchymal stromal cells (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic stage and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. In parallel, IL-33-expressing myelinating Schwann cell progenitors expanded, likely as a repair mechanism for the previously described neuropathy in MPN. IL-33 had anti-fibrotic effects on MSCs but induced expression of the alarmin S100A8 leading to loss-of-hematopoiesis support. Importantly, we demonstrate that S100A8 expression in stroma and patient plasma marks fibrosis progression and might even serve as a biomarker for disease progression.

Ewing´s Sarcoma Family of Tumors (ESFT) are clinically aggressive bone and soft tissue tumors in children and young adults. Analysis of the immune tumor microenvironment (TME) provides insight into tumor evolution and novel treatment options. So far, the scarcity of immune cells in ESFT has hindered a comprehensive analysis of rare subtypes. We determined the relative fraction of 22 immune cell types using 197 microarray gene expression datasets of primary ESFT tumor samples by using CIBERSORT, a deconvolution algorithm enumerating infiltrating leucocytes in bulk tumor tissue. The most abundant cells were macrophages (mean 43% of total tumor-infiltrating leukocytes, TILs), predominantly immunosuppressive M2 type macrophages, followed by T cells (mean 23% of TILs). Increased neutrophils, albeit at low number, were associated with a poor overall survival (OS) (p = .038) and increased M2 macrophages predicted a shorter event-free survival (EFS) (p = .033). High frequency of T cells and activated NK cells correlated with prolonged OS (p = .044 and p = .007, respectively). A small patient population (9/32) with combined low infiltrating M2 macrophages, low neutrophils, and high total T cells was identified with favorable outcome. This finding was confirmed in a validation cohort of patients with follow up (11/38). When comparing the immune TME with expression of known stemness genes, hypoxia-inducible factor 1 α (HIF1α) correlated with high abundance of macrophages and neutrophils and decreased T cell levels. The immune TME in ESFTs shows a distinct composition including rare immune cell subsets that in part may be due to expression of HIF1α.

Aims Despite increasing interest in the recently established immunoglobulin 4-related disease (IgG4-RD), its pathogenesis and aetiology remain largely unclear. Characteristic histopathological features are one of the key elements of diagnosis, including 'storiform' fibrosis, obliterative phlebitis, increased lymphoplasmacytic infiltration and increased levels of IgG4 in serum and tissue. Histopathological features of IgG4-RD are striking but not specific, and can pose a pitfall for surgical pathologists. This paper aims to determine the actual amount of IgG4+ plasma cells in nodular-sclerosing Hodgkin lymphoma (NSHL) and its potential to be misdiagnosed in routine clinical practice. Methods and results IgG4+ plasma cells per high-power field (HPF) and the ratio of IgG4+ versus IgG+ plasma cells (IgG4/IgG ratio) in lymph node biopsies of 24 patients with nodular-sclerosing Hodgkin lymphoma (NSHL) were determined using immunohistochemistry and consensus scoring criteria as used for IgG4-RD. Ten lymph node biopsies with reactive follicular hyperplasia were assessed for comparison. Higher numbers of IgG4+ plasma cells (P < 0.001) were observed in NSHL versus follicular hyperplasia (mean 34 versus 8 per HPF) with a mean IgG4/IgG ratio of 0.38 versus 0.18. Five cases (21%) fulfilled the consensus criteria of IgG4-RD, with >50 IgG4+ plasma cells per HPF and an IgG4/IgG ratio of >0.4. The mean count of IgG4+ plasma cells per HPF in NSHL varied greatly (3-88) with increased numbers of IgG4+ plasma cells seen near areas of fibrosclerosis. Conclusions Significantly higher levels of IgG4+ plasma cells are common in NSHL, emphasising the need to exclude Reed-Sternberg cells by morphology and immunohistochemistry in biopsies where IgG4-RD is suspected.

Im Mittelpunkt des diesjährigen interdisziplinären Symposiums der Arbeitsgemeinschaft (AG) Anämie anlässlich der Jahrestagung der Deutschen Gesellschaft für Geriatrie stand der Vitamin-B12-Mangel im höheren Lebensalter. Experten aus den Bereichen Hämatopathologie, klinische Geriatrie und geriatrische Hämatologie präsentierten gemeinsam den klinischen Fall einer 78-jährigen Patientin und erläuterten auf Basis dieser Kasuistik Epidemiologie, Klinik, Diagnostik und Therapie des Vitamin-B12-Mangels im Alter. Der vorliegende Text gibt unter Berücksichtigung der aktuellen Literatur eine zusammenfassende Übersicht über die Symposiumsbeiträge zum Thema Vitamin-B12-Mangel im Alter.

Anämie im höheren Lebensalter ist ein multifaktorielles Problem, das nach einer interdisziplinären Herangehensweise verlangt. Die Beiträge dieses ersten interdisziplinären Symposiums zu Anämie im höheren Lebensalter umfassten physiologische, histopathologische, nephrologische und neurogeriatrische Aspekte sowie therapeutische Optionen für diese häufige, jedoch noch immer unterdiagnostizierte Erkrankung. Das Symposium war die Auftaktveranstaltung zur Neugründung der Arbeitsgemeinschaft Anämie der Deutschen Gesellschaft für Geriatrie.