High-dose chemotherapy and autologous haematopoietic stem-cell transplantation in older, fit patients with primary diffuse large B-cell CNS lymphoma (MARTA): a single-arm, phase 2 trial.

BACKGROUND: Available treatments for older patients with primary diffuse large B-cell CNS lymphoma (PCNSL) offer progression-free survival of up to 16 months. We aimed to investigate an intensified treatment of high-dose chemotherapy and autologous haematopoietic stem-cell transplantation (HSCT) in older patients with PCNSL. METHODS: MARTA was a prospective, single-arm, phase 2 study done at 15 research hospitals in Germany. Patients aged 65 years or older with newly diagnosed, untreated PCNSL were enrolled if they had an Eastern Cooperative Oncology Group performance status of 0-2 and were fit for high-dose chemotherapy and autologous HSCT. Induction treatment consisted of two 21-day cycles of high-dose intravenous methotrexate 3·5 g/m2 (day 1), intravenous cytarabine 2 g/m2 twice daily (days 2 and 3), and intravenous rituximab 375 mg/m2 (days 0 and 4) followed by high-dose chemotherapy with intravenous rituximab 375 mg/m2 (day -8), intravenous busulfan 3·2 mg/kg (days -7 and -6), and intravenous thiotepa 5 mg/kg (days -5 and -4) plus autologous HSCT. The primary endpoint was progression-free survival at 12 months in all patients who met eligibility criteria and started treatment. The study was registered with the German clinical trial registry, DRKS00011932, and recruitment is complete. FINDINGS: Between Nov 28, 2017, and Sept 16, 2020, 54 patients started induction treatment and 51 were included in the full analysis set. Median age was 71 years (IQR 68-75); 27 (53%) patients were female and 24 (47%) were male. At a median follow-up of 23·0 months (IQR 16·8-37·4), 23 (45%) of 51 patients progressed, relapsed, or died. 12-month progression-free survival was 58·8% (80% CI 48·9-68·2; 95% CI 44·1-70·9). During induction treatment, the most common grade 3-5 toxicities were thrombocytopenia and leukopenia (each in 52 [96%] of 54 patients). During high-dose chemotherapy and autologous HSCT, the most common grade 3-5 toxicity was leukopenia (37 [100%] of 37 patients). Treatment-related deaths were reported in three (6%) of 54 patients, all due to infectious complications. INTERPRETATION: Although the primary efficacy threshold was not met, short induction followed by high-dose chemotherapy and autologous HSCT is active in selected older patients with PCNSL and could serve as a benchmark for comparative trials. FUNDING: Else Kröner-Fresenius Foundation, Riemser Pharma, and Medical Center-University of Freiburg.

A prospective observational study of real-world treatment and outcome in secondary CNS lymphoma.

BACKGROUND: Secondary central nervous system lymphoma (SCNSL) confers a dismal prognosis and treatment advances are constrained by the lack of prospective studies and real-world treatment evidence. METHODS: Patients with SCNSL of all entities were included at first diagnosis and patient characteristics, treatment data, and outcomes were prospectively collected in the Secondary CNS Lymphoma Registry (SCNSL-R) (NCT05114330). FINDINGS: 279 patients from 47 institutions were enrolled from 2011 to 2022 and 243 patients (median age: 66 years; range: 23-86) were available for analysis. Of those, 49 (20 %) patients presented with synchronous (cohort I) and 194 (80 %) with metachronous SCNSL (cohort II). The predominant histology was diffuse large B-cell lymphoma (DLBCL, 68 %). Median overall survival (OS) from diagnosis of CNS involvement was 17·2 months (95 % CI 12-27·5), with longer OS in cohort I (60·6 months, 95 % CI 45·5-not estimable (NE)) than cohort II (11·4 months, 95 % CI 7·8-17·7, log-rank test p < 0.0001). Predominant induction regimens included R-CHOP/high-dose MTX (cohort I) and high-dose MTX/cytarabine (cohort II). Rituximab was used in 166 (68 %) of B-cell lymphoma. Undergoing consolidating high-dose therapy and autologous hematopoietic stem cell transplantation (HDT-ASCT) in partial response (PR) or better was associated with longer OS (HR adjusted 0·47 (95 % CI 0·25-0·89), p = 0·0197). INTERPRETATION: This study is the largest prospective cohort of SCNSL patients providing a comprehensive overview of an international real-world treatment landscape and outcomes. Prognosis was better in patients with SCNSL involvement at initial diagnosis (cohort I) and consolidating HDT-ASCT was associated with favorable outcome in patients with PR or better.

Patient Reported and Clinical Outcomes after High-Dose Chemotherapy and Autologous Stem Cell Transplantation in Primary Central Nervous System Lymphoma.

Primary central nervous system lymphomas (PCNSL) are rare and associated with an adverse prognosis. High-dose chemotherapy followed by autologous stem cell transplantation (HDC/ASCT) improves progression free (PFS) and overall survival (OS) but neurocognition, performance status and quality of life (QoL) in patient-reported outcome (PRO) after HDC/ASCT remains underexplored. Especially elderly patients may insufficiently recover from this demanding therapy. Therefore, this single-center analysis investigated all PCNSL patients who received HDC/ASCT at the University Hospital Tübingen from 2006-2021 (n = 40, median age 60.5 years) in a retrospective manner. The 2-year PFS/OS was 78.7%/77.3%, respectively, without significant differences between the tested age-groups (≤60 vs. >60 years, p = 0.531/p = 0.334). Higher Thiotepa dosage was an independent predictor for better OS (p = 0.018). Additionally, a one-time prospective, cross-sectional analysis after HDC/ASCT in the same cohort was performed (n = 31; median follow-up 45 months). Here, the median ECOG improved by HDC/ASCT from 1 to 0 and mini-mental state examinations revealed unimpaired neurocognitive functioning (median 28 pts.). PRO data collected by EORTC QLQ-C30 showed a good QoL in both age groups with an average global health status (GHS) of 68.82% (≤60y: 64.72%, >60y: 74.14%). Together, our data indicate that HDC/ASCT is an effective therapy with respect to disease control, overall health status and quality of life, irrespective of patient age.

ALK-positive histiocytosis: a new clinicopathologic spectrum highlighting neurologic involvement and responses to ALK inhibition.

ALK-positive histiocytosis is a rare subtype of histiocytic neoplasm first described in 2008 in 3 infants with multisystemic disease involving the liver and hematopoietic system. This entity has subsequently been documented in case reports and series to occupy a wider clinicopathologic spectrum with recurrent KIF5B-ALK fusions. The full clinicopathologic and molecular spectra of ALK-positive histiocytosis remain, however, poorly characterized. Here, we describe the largest study of ALK-positive histiocytosis to date, with detailed clinicopathologic data of 39 cases, including 37 cases with confirmed ALK rearrangements. The clinical spectrum comprised distinct clinical phenotypic groups: infants with multisystemic disease with liver and hematopoietic involvement, as originally described (Group 1A: 6/39), other patients with multisystemic disease (Group 1B: 10/39), and patients with single-system disease (Group 2: 23/39). Nineteen patients of the entire cohort (49%) had neurologic involvement (7 and 12 from Groups 1B and 2, respectively). Histology included classic xanthogranuloma features in almost one-third of cases, whereas the majority displayed a more densely cellular, monomorphic appearance without lipidized histiocytes but sometimes more spindled or epithelioid morphology. Neoplastic histiocytes were positive for macrophage markers and often conferred strong expression of phosphorylated extracellular signal-regulated kinase, confirming MAPK pathway activation. KIF5B-ALK fusions were detected in 27 patients, whereas CLTC-ALK, TPM3-ALK, TFG-ALK, EML4-ALK, and DCTN1-ALK fusions were identified in single cases. Robust and durable responses were observed in 11/11 patients treated with ALK inhibition, 10 with neurologic involvement. This study presents the existing clinicopathologic and molecular landscape of ALK-positive histiocytosis and provides guidance for the clinical management of this emerging histiocytic entity.

Imatinib dose reduction in major molecular response of chronic myeloid leukemia: results from the German Chronic Myeloid Leukemia-Study IV.

Standard first-line therapy of chronic myeloid leukemia is treatment with imatinib. In the randomized German Chronic Myeloid Leukemia-Study IV, more potent BCR-ABL inhibition with 800 mg ('high-dose') imatinib accelerated achievement of a deep molecular remission. However, whether and when a de-escalation of the dose intensity under high-dose imatinib can be safely performed without increasing the risk of losing deep molecular response is unknown. To gain insights into this clinically relevant question, we analyzed the outcome of imatinib dose reductions from 800 mg to 400 mg daily in the Chronic Myeloid Leukemia-Study IV. Of the 422 patients that were randomized to the 800 mg arm, 68 reduced imatinib to 400 mg after they had achieved at least a stable major molecular response. Of these 68 patients, 61 (90%) maintained major molecular remission on imatinib at 400 mg. Five of the seven patients who lost major molecular remission on the imatinib standard dose regained major molecular remission while still on 400 mg imatinib. Only two of 68 patients had to switch to more potent kinase inhibition to regain major molecular remission. Importantly, the lengths of the intervals between imatinib high-dose treatment before and after achieving major molecular remission were associated with the probabilities of maintaining major molecular remission with the standard dose of imatinib. Taken together, the data support the view that a deep molecular remission achieved with high-dose imatinib can be safely maintained with standard dose in most patients. Study protocol registered at clinicaltrials.gov 00055874.

Phase I Study of Oncolytic Vaccinia Virus GL-ONC1 in Patients with Peritoneal Carcinomatosis.

Purpose: Peritoneal carcinomatosis is common in advanced tumor stages or disease recurrence arising from gastrointestinal cancers, gynecologic malignancies, or primary peritoneal carcinoma. Because current therapies are mostly ineffective, new therapeutic approaches are needed. Here, we report on a phase I study designed to assess safety, MTD, and antitumor activity of intraperitoneal administration of oncolytic vaccinia virus GL-ONC1 in advanced stage peritoneal carcinomatosis patients.Patients and Methods: GL-ONC1 was administered intraperitoneally every 4 weeks for up to four cycles at three different dose levels (107-109 pfu) following a standard 3+3 dose escalation design. GL-ONC1 was infused via an indwelling catheter that enabled repetitive analyses of peritoneal fluid biopsies. The primary study objective was safety of GL-ONC1 according to Common Terminology Criteria for Adverse Events, version 4.0 (CTCAEv4.0).Results: Patients with advanced-stage peritoneal carcinomatosis (n = 7) or advanced peritoneal mesothelioma (n = 2) received 24 doses of GL-ONC1. Adverse events were limited to grades 1-3, including transient flu-like symptoms and increased abdominal pain, resulting from treatment-induced peritonitis. No DLT was reported, and the MTD was not reached. Furthermore, no signs of viral shedding were observed. Importantly, in 8 of 9 study patients, effective intraperitoneal infections, in-patient replication of GL-ONC1, and subsequent oncolysis were demonstrated in cycle 1. All patients developed neutralizing activities against GL-ONC1.Conclusions: GL-ONC1 was well tolerated when administered into the peritoneal cavity of patients with advanced stage peritoneal carcinomatosis. Efficient tumor cell infection, in-patient virus replication, and oncolysis were limited to treatment cycle 1 (ClinicalTrials.gov number, NCT01443260). Clin Cancer Res; 24(18); 4388-98. ©2018 AACR.

High-dose chemotherapy with autologous haemopoietic stem cell transplantation for newly diagnosed primary CNS lymphoma: a prospective, single-arm, phase 2 trial.

BACKGROUND: High-dose methotrexate-based chemotherapy is standard for primary CNS lymphoma, but most patients relapse. High-dose chemotherapy with autologous stem cell transplantation (HCT-ASCT) is supposed to overcome the blood-brain barrier and eliminate residual disease in the CNS. We aimed to investigate the safety and efficacy of HCT-ASCT in patients with newly diagnosed primary CNS lymphoma. METHODS: In this prospective, single-arm, phase 2 trial, we recruited patients aged 18-65 years with newly diagnosed primary CNS lymphoma and immunocompetence, with no limitation on clinical performance status, from 15 hospitals in Germany. Patients received five courses of intravenous rituximab 375 mg/m(2) (7 days before first high-dose methotrexate course and then every 10 days) and four courses of intravenous high-dose methotrexate 8000 mg/m(2) (every 10 days) and then two courses of intravenous rituximab 375 mg/m(2) (day 1), cytarabine 3 g/m(2) (days 2 and 3), and thiotepa 40 mg/m(2) (day 3). 3 weeks after the last course, patients commenced intravenous HCT-ASCT (rituximab 375 mg/m(2) [day 1], carmustine 400 mg/m(2) [day 2], thiotepa 2 × 5 mg/kg [days 3 and 4], and infusion of stem cells [day 7]), irrespective of response status after induction. We restricted radiotherapy to patients without complete response after HCT-ASCT. The primary endpoint was complete response at day 30 after HCT-ASCT in all registered eligible patients who received at least 1 day of study treatment. This trial is registered at ClinicalTrials.gov, number NCT00647049. FINDINGS: Between Jan 18, 2007, and May 23, 2011, we recruited 81 patients, of whom two (2%) were excluded, therefore we included 79 (98%) patients in the analysis. All patients started induction treatment; 73 (92%) commenced HCT-ASCT. 61 (77·2% [95% CI 66·1-86·6]) patients achieved a complete response. During induction treatment, the most common grade 3 toxicity was anaemia (37 [47%]) and the most common grade 4 toxicity was thrombocytopenia (50 [63%]). During HCT-ASCT, the most common grade 3 toxicity was fever (50 [68%] of 73) and the most common grade 4 toxicity was leucopenia (68 [93%] of 73). We recorded four (5%) treatment-related deaths (three [4%] during induction and one [1%] 4 weeks after HCT-ASCT). INTERPRETATION: HCT-ASCT with thiotepa and carmustine is an effective treatment option in young patients with newly diagnosed primary CNS lymphoma, but further comparative studies are needed. FUNDING: University Hospital Freiburg and Amgen.

Allogeneic hematopoietic cell transplantation in patients with myelofibrosis: A single center experience.

Myelofibrosis (MF) is a rare disease responsible for an increasing ineffective hematopoesis by a progressive fibrosing process in the bone marrow. The only curative treatment option is allogeneic hematopoietic cell transplantation (HCT). In this single-center analysis, we evaluated retrospectively 54 consecutive patients suffering from primary or secondary MF which underwent HCT from 1997 to 2014 after either myeloablative (MAC, n = 19) or reduced-intensity conditioning (RIC, n = 35). Overall survival (OS) and disease-free survival (DFS) after 3 years was 54/53 % for RIC versus 63/58 % for MAC (p = 0.8/0.97). Cumulative incidence of relapse was 34 % after RIC and 8 % after MAC (p = 0.16). Three-year non-relapse mortality (NRM) was 15 % after RIC and 34 % after MAC (p = 0.29). We found that RIC was associated with a lower incidence of acute graft versus host disease (GvHD; II-IV 26 vs. 0 %, p = 0.004). Evaluation of prognostic relevance of the Dynamic International Prognostic Scoring System (DIPSS) score showed a significant better OS in patient with risk score ≤3 versus >3 (after 3 years, 71 vs. 39 %, p = 0.008). While similar OS and DFS were observed with MAC or RIC, the use of RIC resulted in lower incidence of acute GvHD. RIC regimens may be therefore the preferred conditioning approach for allogeneic HCT in patients with MF.

Prognostic relevance of HER2/neu in acute lymphoblastic leukemia and induction of NK cell reactivity against primary ALL blasts by trastuzumab.

The epidermal growth factor receptor HER2/neu is expressed on various cancers and represents a negative prognostic marker, but is also a target for the therapeutic monoclonal antibody Trastuzumab. In about 30% of cases, HER2/neu is expressed on acute lymphoblastic leukemia (ALL) cells and was proposed to be associated with a deleterious prognosis. Here we evaluated clinical data from 65 ALL patients (HER2/neu+, n = 17; HER2/neu-, n = 48) with a median follow-up of 19.4 months (range 0.6-176.5 months) and observed no association of HER2/neu expression with response to chemotherapy, disease free or overall survival. In vitro, treatment of primary ALL cells (CD20+HER2/neu+, CD20+HER2/neu- and CD20-HER2/neu-) with Rituximab and Trastuzumab led to activation of NK cells in strict dependence of the expression of the respective antigen. NK reactivity was more pronounced with Rituximab as compared to Trastuzumab, and combined application could lead to additive effects in cases where both antigens were expressed. Besides providing evidence that HER2/neu expression is no risk factor in ALL patients, our data demonstrates that HER2/neu can be a promising target for Trastuzumab therapy in the subset of ALL patients with the potential to improve disease outcome.

Randomized phase III study of whole-brain radiotherapy for primary CNS lymphoma.

OBJECTIVE: This is the final report of a phase III randomized study to evaluate whole-brain radiotherapy (WBRT) in primary therapy of primary CNS lymphoma (PCNSL) after a median follow-up of 81.2 months. METHODS: Patients with newly diagnosed PCNSL were randomized to high-dose methotrexate (HDMTX)-based chemotherapy alone or followed by WBRT. We hypothesized that the omission of WBRT would not compromise overall survival (OS; primary endpoint), using a noninferiority design with a margin of 0.9. RESULTS: In the per-protocol population (n = 320), WBRT nonsignificantly prolonged progression-free survival (PFS) (median 18.2 vs 11.9 months, hazard ratio [HR] 0.83 [95% confidence interval (CI) 0.65-1.06], p = 0.14) and significantly PFS from last HDMTX (25.5 vs 12.0 months, HR 0.65 [95% CI 0.5-0.83], p = 0.001), but without OS prolongation (35.6 vs 37.1 months, HR 1.03 [95% CI 0.79-1.35], p = 0.82). In the intent-to-treat population (n = 410), there was a prolongation by WBRT of both PFS (15.4 vs 9.9 months, HR 0.79 [95% CI 0.64-0.98], p = 0.034) and PFS from last HDMTX (19.4 vs 11.9 months, HR 0.72 [95% CI 0.58-0.89], p = 0.003), but not of OS (32.4 vs 36.1 months, HR 0.98 [95% CI 0.79-1.26], p = 0.98). CONCLUSION: Although the statistical proof of noninferiority regarding OS was not given, our results suggest no worsening of OS without WBRT in primary therapy of PCNSL. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that in PCNSL HDMTX-based chemotherapy followed by WBRT does not significantly increase survival compared to chemotherapy alone. The study lacked the precision to exclude an important survival benefit or harm from WBRT.

Successful use of eculizumab for treatment of an acute hemolytic reaction after ABO-incompatible red blood cell transfusion.

BACKGROUND: Transfusion of ABO major-incompatible red blood cells (RBCs) can activate the complement system and can cause severe and even lethal acute hemolytic reactions. The activation of the complement system with formation of C3a and C5a (anaphylatoxins) and the release of hemoglobin from the lysed RBCs are thought to mediate clinical signs like fever, hypotension, pain, and acute renal failure. Therapeutic inhibition of the complement cascade in case of ABO-incompatible RBC transfusion would be desirable to ameliorate the signs and symptoms and to improve the outcome of the reaction. STUDY DESIGN AND METHODS: A patient with blood group B was erroneously transfused with a unit of group A2 RBCs. Within 1 hour after transfusion she received eculizumab, a monoclonal antibody that binds to the complement component C5 and blocks its cleavage. Clinical and immunohematologic observations are reported here. RESULTS: Hemoglobinemia and hemoglobinuria were present for several hours after transfusion, but she developed no hypotension, no renal failure, and no disseminated intravascular coagulation. As shown by flow cytometry, group A cells survived in the peripheral blood for more than 75 days. No immunoglobulin G was detectable by column agglutination technique on these cells. CONCLUSION: A low isoagglutinin titer and blood group A2 of the erroneously transfused cells most likely were the reason for the absence of clinical signs during and immediately after the ABO-incompatible transfusion. In the further course, eculizumab successfully protected the incompatible RBCs from hemolysis for several weeks.

Phase II study of central nervous system (CNS)-directed chemotherapy including high-dose chemotherapy with autologous stem cell transplantation for CNS relapse of aggressive lymphomas.

The prognosis of patients with central nervous system relapse of aggressive lymphoma is very poor with no therapy established so far. In a prospective multicenter phase II study, we evaluated a potentially curative chemotherapy-only regimen in these patients. Adult immunocompetent patients 65 years of age or under received induction chemotherapy with MTX/IFO/DEP (methotrexate 4 g/m(2) intravenously (i.v.) Day 1, ifosfamide 2 g/m(2) i.v. Days 3- 5 and liposomal cytarabine 50 mg intrathecally (i.th) Day 6) and AraC/TT/DEP (cytarabine 3g/m(2) i.v. Days 1-2, thiotepa 40 mg/m(2) i.v. Day 2 and i.th. liposomal cytarabine 50 mg i.th. Day 3) followed by high-dose chemotherapy with carmustine 400 mg/m(2) i.v. Day -5, thiotepa 2×5 mg/kg i.v. Days -4 to -3 and etoposide 150 mg/m(2) i.v. Days -5 to -3, and autologous stem cell transplantation Day 0 (HD-ASCT). Thirty eligible patients (median age 58 years) were enrolled. After HD-ASCT (n=24), there was a complete remission in 15 (63%), partial remission in 2 (8%) and progressive disease in 7 (29%) patients. Myelotoxicity was the most adverse event with CTC grade 3/4 infections in 12% of MTX/IFO/DEP courses, 21% of AraC/TT/DEP courses and 46% of HD-ASCT courses. The 2-year time to treatment failure was 49%±19 for all patients and 58%±22 for patients completing HD-ASCT. The protocol assessed proved feasible and highly active with long-lasting remissions in a large proportion of patients. (ClinicalTrials.govIdentifier NCT01148173).

Outcome of elderly patients with primary CNS lymphoma in the G-PCNSL-SG-1 trial.

OBJECTIVE: To assess the outcome of elderly patients with primary CNS lymphoma (PCNSL) treated within the G-PCNSL-SG-1 trial. METHODS: We reviewed response, toxicity, and survival of patients with PCNSL aged 70 or more enrolled in the G-PCNSL-SG-1 trial. RESULTS: A total of 126 of the 526 eligible patients (24%) and 66 of 318 patients (21%) in the per protocol population were aged 70 or more. Among all eligible patients, the rate of complete and partial responses (CR+PR) to HD-MTX-based chemotherapy was 44% in the elderly vs 57% in the younger patients (p = 0.016). Toxicity was age-independent except for a higher rate of grade III/IV leukopenia in the elderly (34% vs 21%, p = 0.007). Death on therapy was more frequent (18% vs 11%; p = 0.027), and progression-free survival (PFS) (4.0 vs 7.7 months, p = 0.014) and overall survival (12.5 vs 26.2 months, p < 0.001) inferior, in the elderly. A striking difference between younger and elderly patients was the PFS of CR patients of 35.0 in the younger vs 16.1 in the elderly patients (p = 0.024). Elderly patients were treated less often and less aggressively at salvage. However, age was not associated with survival from salvage whole brain radiotherapy in patients progressing during primary HD-MTX-based chemotherapy (p = 0.633). CONCLUSIONS: Lower response rate and higher mortality on HD-MTX-based chemotherapy as well as lower PFS of CR patients and less salvage therapy contribute to the poor prognosis of elderly patients with PCNSL.

G protein-coupled receptor crosstalk and signaling in hematopoietic stem and progenitor cells.

A variety of G protein-coupled receptors (GPCRs) is expressed in hematopoietic stem and progenitor cells (HPCs), including the chemokine receptor CXCR4, the leukotriene receptor CysLT1, the sphingosine 1-phosphate receptor S1P1, the cannabinoid receptor CB2, and the complement receptor C3aR. While the role of CXCR4 in stem cell homing is largely established, the function of the other GPCRs expressed in HPCs is only partially understood. CXCR4 and CysLT1 inhibit their own activation after ligand binding (homologous desensitization). Stimulation of S1P1 or C3aR has been shown to activate CXCR4 in HPCs that may sensitize CXCR4-dependent stem cell homing. In contrast, activation of CXCR4 results in a loss of CysLT1 function, which is most likely mediated by protein kinase C (PKC) signaling (heterologous desensitization) and could explain the ineffectiveness of CysLT1 antagonists to mobilize HPCs in vivo. Further characterization of GPCR crosstalk will allow a better understanding of HPC trafficking.

Osteoprotegerin positively regulates hematopoietic progenitor cells.

Osteoprotegerin (OPG), the soluble decoy receptor of RANKL is released by bone marrow osteoblasts and plays an important role in physiological osteoblastogenesis and pathological bone disease. In earlier studies, we have shown that generated stromal cell lines from the aorta-gonad-mesonephros (AGM)-region serving as good supporters of murine and human hematopoietic progenitor cell (HPC) expansion highly express OPG detected by microarray analysis. Here, we investigated the role of OPG to HPC expansion in vitro. Addition of OPG leads to an enhanced expansion of HPC in liquid culture. In addition, progenitor cell function, measured by colony and cobblestone formation, was increased. The observed effects were partially antagonized by addition of RANKL. In conclusion, these findings suggest an important role of OPG maintaining progenitor cell function in the osteoblastic niche.

The G protein-coupled receptor CysLT1 mediates chemokine-like effects and prolongs survival in chronic lymphocytic leukemia.

The G protein-coupled receptor (GPCR) CXCR4 is involved in bone marrow tropism and survival of chronic lymphocytic leukemia (CLL) cells. The function of the GPCRs cysteinyl leukotriene receptor 1 (CysLT1) and CysLT2 remains elusive. Here we demonstrate that in CLL and normal B lymphocytes, CysLT1 mRNA is consistently expressed, in contrast to low CysLT2 levels. Similar to the CXCR4 ligand CXCL12, the cysteinyl leukotriene (cysLT) LTD(4) induces calcium fluxes, actin polymerization, and chemotaxis. These effects are blocked by specific CysLT1 antagonists. Their inhibition by pertussis toxin suggests Giα/o protein involvement. Furthermore, CysLT1 mediates MAP-kinase phosphorylation, which implicates contribution of cysLT to survival. Indeed, CysLT1 antagonists induce apoptosis and reduce viability independent of Gαi/o protein signaling. Considering the production of cysLTs in the bone marrow, our data suggest that CysLT1 induces chemokine-like effects, supports accumulation and survival of CLL cells in the bone marrow and thus represents a potential treatment target.

Glioma tropism of lentivirally transduced hematopoietic progenitor cells.

Experimental gliomas attract hematopoietic progenitor cells (HPC) in vivo. HPC are therefore promising candidates for a cell-based delivery of therapeutic molecules to experimental gliomas. A therapeutic application requires efficient genetic manipulation of the cellular vector and a lack of tumorigenicity. Here, we studied the impact of lenti-viral transduction on the glioma tropism of human or murine HPC. Transduction of human or murine HPC with a GFP lentivirus (lenti-GFP) did not interfere with the glioma-mediated attraction of HPC. Bone marrow reconstitution of C57Bl/6 mice with syngeneic GFP-transgenic lineage-depleted bone marrow cells (lin- BM) was as efficient as reconstitution with syngeneic lin- BM transduced ex vivo with lenti-GFP. SMA-560 gliomas growing orthotopically in lenti-GFP-reconstituted VM/Dk mice recruited GFP-positive bone marrow-derived cells. Thus, lentiviral transduction did not interfere with the attraction of exogenously injected HPC or endogenous bone marrow-derived cells by experimental gliomas. Lenti-GFP-HPC implanted directly into tumor-free brains were not tumorigenic. The intravenous injection of lenti-GFP-HPC in glioma-bearing mice did not alter the survival of otherwise untreated animals and had no impact on the survival benefit conferred by cerebral irradiation. Taken together, genetic manipulation of HPC with lenti-GFP neither made these cells tumorigenic nor interfered with their glioma tropism.

In vitro myogenic differentiation of human bone marrow-derived mesenchymal stem cells as a potential treatment for urethral sphincter muscle repair.

To explore a new treatment strategy for urinary incontinence, human bone marrow mesenchymal stem cells (MSC) of the first in vitro passage were exposed to 5-azacytidine (AZA) to induce myogenic differentiation, and cultured for a total of six passages. Expression of stem cell surface antigens and intracellular alpha-actin was examined by flow cytometry at the end of each passage and compared to that of native MSC (not exposed to AZA) cultured in parallel. To analyze differentiation into striated muscle, expression of the transcription factor MyoD1 and myosin heavy chain (MyHC) was examined by RT-PCR. Both native and AZA-exposed MSC of all passages were negative for the progenitor/endothelial antigen CD34, leukocytic CD45, and endothelial/monocytic CD31. In contrast, the MSC markers CD73, CD90, CD105, and intracellular actin were detected in both groups of MSC throughout the culture period. After an initial increase, the expression level of MSC antigens decreased over time particularly in AZA-exposed MSC. Expression of smooth muscle alpha-actin also declined, but was greater in AZA-exposed MSC throughout the culture period. Varying percentages of MSC cultures expressed MyoD1 and MyHC mRNA. In late passages, AZA-exposed MSC tended to be more frequently positive than native MSC. In pilot experiments, transplantation of MSC into the bladder neck tissue of athymic rats was feasible; long-term analyses are pending. We conclude that independent of AZA exposure, MSC express smooth and striated muscle antigens. Treatment with AZA slightly increases myogenic differentiation, but may not be necessary in future studies of MSC as a treatment modality for urinary incontinence.

The CysLT1 ligand leukotriene D4 supports alpha4beta1- and alpha5beta1-mediated adhesion and proliferation of CD34+ hematopoietic progenitor cells.

Cytokines and chemokines control hematopoietic stem and progenitor cell (HPC) proliferation and trafficking. However, the role of nonpeptide mediators in the bone marrow microenvironment has remained elusive. Particularly CysLT(1), a G protein-coupled receptor recognizing inflammatory mediators of the cysteinyl leukotriene family, is highly expressed in HPCs. We therefore analyzed the effects of its ligands on human CD34(+) HPCs. The most potent CysLT(1) ligand, LTD(4), rapidly and significantly up-regulated alpha(4)beta(1) and alpha(5)beta(1) integrin-dependent adhesion of both primitive and committed HPC. LTD(4)-triggered adhesion was inhibited by specific CysLT(1) antagonists. The effects of other CysLT(1) ligands were weak (LTC(4)) or absent (LTE(4)). In serum-free liquid cultures supplemented with various hematopoietic cytokines including IL-3, only LTD(4) significantly augmented the expansion of HPCs in a dose-dependent manner comparable to that of peptide growth factors. LTC(4) and LTE(4) were less effective. In CD34(+) cell lines and primary HPCs, LTD(4) induced phosphorylation of p44/42 ERK/MAPK and focal adhesion kinase-related tyrosine kinase Pyk2, which is linked to integrin activation. Bone marrow stromal cells produced biologically significant amounts of cysteinyl leukotrienes only when hematopoietic cells were absent, suggesting a regulatory feedback mechanism in the hematopoietic microenvironment. In contrast to antagonists of the homing-related G protein-coupled receptor CXCR4, administration of a CysLT(1) antagonist failed to induce human CD34(+) HPC mobilization in vivo. Our results suggest that cysteinyl leukotriene may contribute to HPC retention and proliferation only when cysteinyl leukotriene levels are increased either systemically during inflammation or locally during marrow aplasia.

Modulation of CXC chemokine receptor expression and function in human neutrophils during aging in vitro suggests a role in their clearance from circulation.

In mice, differential regulation of CXC chemokine receptor expression in circulating polymorphonuclear neutrophils (PMNs) undergoing senescence results in homing to the bone marrow. However, the role of this compartment and of the chemokine receptor CXCR4 is still under discussion, and only scarce data exist about CXCR4 function in human PMN. In our study, we provide evidence that also in human neutrophils, expression (cell surface and mRNA), chemotactic and signaling functions of the homing-related chemokine receptor CXCR4 are upregulated during aging in vitro, independent of addition of stimulatory cytokines (TNF, IL-1, IL-8, G-CSF). In contrast, interleukin-8 receptors are downmodulated (CXCR2) or remain unchanged (CXCR1), suggesting that human PMNs undergoing senescence acquire a phenotype that impairs inflammatory extravasation and favors homing to the bone marrow or other tissues involved in sequestration. Partially retained responsiveness to interleukin-8 may be important for neutrophil function when senescence occurs after extravasation in inflamed tissues.