bluuebunny/crossref_metadata_2025 huggingface dataset:
Preclinical Efficacy of Campthothecin-Polymer Conjugate (IT-101) in Human Burkitt Lymphoma Bearing Mice.
Authors: Numbenjapon Tontanai , Wang Jianyi , Colcher David , Schluep Thomas , Forman Stephen J. , Raubitschek Andrew
Published: October 2019

First line combination chemotherapy can result in long-term disease-free survival (DFS) in 65–90% of patients with Burkitt lymphoma (BL) depending on prognostic factors at presentation. However, only a small number of patients with relapsed/refractory diseases can achieve long-term DFS after the high-dose therapy and hematopoietic stem cell rescue. Irinotecan (CPT-11) and topotecan, two analogues of campthothecin (CPT), have been successfully used alone or in combination with other cytotoxic agents as salvage treatments for patients with relapsed/refractory non-Hodgkin’s lymphoma (NHL) other than BL. A disadvantage of CPT-11 and topotecan observed in early clinical trials was a high incidence of treatment-related toxicity (TRT). IT-101, a nanoparticulate conjugate of 20(S)-camptothecin and a cyclodextrin-based polymer, has recently been developed to minimize TRT and increase anti-tumor activity. To evaluate preclinical efficacy and toxicity of IT-101, we conducted an in vivo study in nude mice bearing subcutaneous xenografts from human BL-derived Daudi cell line stably transfected with plasmid DNA containing firefly luciferase (ffluc) reporter gene. The tumor-derived ffluc activity was evaluated in vivo using an IVIS 100 imaging system (Xenogen); background ffluc luminescence was defined as ≤106 p/sec/cm2/sr. Ten days after subcutaneous tumor injection, 42 nude mice with tumor-derived ffluc activity of >108 p/sec/cm2/sr were allocated into six different treatment groups as followings: Group 1 (no therapy); Group 2 (CPT-11 100mg/kg, ip, qwk × 3); Group 3 (IT-101 5mg/kg, iv, qwk × 3); Group 4 (IT-101 10mg/kg, iv, qwk × 3); Group 5 (IT-101 15mg/kg, iv, qwk × 2); and Group 6 (IT-101 single dose of 15mg/kg, iv). The average tumor-derived ffluc activity among all of the treatment groups was equally distributed. Tumor burden was monitored weekly by tumor-derived ffluc activity and physical tumor measurements. After completion of the treatment, there were two treatment-related deaths (TRDs; body weight loss >20%) and one non-TRD in group 5 and 6, respectively. At the 3rd week after starting therapy, all treatment groups that received two or three weekly doses of IT-101 (Groups 3, 4, and 5) had 100% complete tumor regression (CTR), determined by ffluc activity (<106 p/sec/cm2/sr) and remained tumor free at 90 days post-therapy. In contrast, 57% (4/7) of mice in Group 2 and 83% (5/6) of mice in Group 6 had initial CTR but all had tumor re-growth by day 69. These results demonstrate that a single dose of IT-101 at 15mg/kg is not sufficiently efficacious while two weekly doses of IT-101 at 15mg/kg are associated with unacceptable toxicity. However, we are able to demonstrate that the three weekly doses of IT-101 at 5 or 10mg/kg are well tolerated and can produce superior anti-tumor activity against Daudi cell line as compared with CPT-11.
Mechanistic Evaluation of Siplizumab (MEDI-507) Activity on Normal and Malignant T-Lymphocytes.
Authors: Hammond Scott A. , Fuhrmann Stacy , Roff Shannon , Kiener Peter A. , Coats Steve , Kinch Michael S.
Published: October 2019

Siplizumab is a humanized monoclonal antibody that selectively binds human CD2 (huCD2), which is expressed on virtually all cells of the T- or NK-cell lineage. Also, known as MEDI-507, siplizumab is being developed as a potential new treatment for lymphoid malignancies involving T- or NK-cells. Our present study was designed to increase further the mechanistic understanding of siplizumab activity. Preclinical studies demonstrated that siplizumab mediated efficient lysis of malignant T-cells in a concentration-dependent and effector-to-target cell ratio-dependent manner. ADCC contributed prominently to tumor cell clearance whereas complement-mediated killing (CDC) did not. Siplizumab treatment alone was sufficient to trigger limited internalization of its target antigen (CD2) from the surface of tumor cells. Moreover, the efficiency of CD2 internalization was increased when such studies were carried out in the presence of an activated monocyte cell line (THP-1) bearing Fcg receptors and incapable of mediating ADCC, and suggested that CD2 clearance from the cell surface can be distinguished from host effector mechanisms. CD2 has been long known to confer both inhibitory and stimulatory signals and we reasoned that target internalization might disrupt survival signals independent of host effector activity. Consistent with this concept, siplizumab treatment in the absence of effector cells transduced apoptotic signals as indicated by multiple standard markers of tumor cell apoptosis , including activation of caspase 3 activity and an increase in tumor cells with a sub-G1 DNA content. In addition, the use of transgenic mouse models demonstrated that intravenous administration of siplizumab caused a rapid (≤ 24 hours) but transient reduction in the number of peripheral huCD2-positive T-cells. The number of T-cells gradually recovered to normal levels within 30 days of treatment. In contrast, siplizumab treatment did not alter the number of CD19-positive B-cells. These results indicate that siplizumab can utilize host effector mechanisms (ADCC) to facilitate malignant T-cell elimination. Our present findings also suggest that siplizumab can mediate changes in CD2 expression or function that result in apoptosis of CD2-positive tumor cells. In light of this potent anti-tumor activity, two Phase I dose escalation trials (MI-CP099 and MI-CP107) are ongoing to assess the maximum tolerated dose and safety of siplizumab in patients with CD2-positive T- and NK-cell malignancies.
Prenylation Inhibitors as Potential Therapeutic Drugs in Hodgkin Lymphoma.
Authors: von Tresckow Bastian , Langendorf Peer , von Strandmann Elke Pogge , Sasse Stefanie , Tawadros Samir , Engert Andreas , Hansen Hinrich P.
Published: October 2019

Although the therapy of Hodgkin lymphoma has improved, patients still suffer from late toxicities and insufficient treatment of relapses. Hence, new treatment strategies are needed. Statins are used to treat hypercholesterolaemia and prevent cardiovascular disease. There is evidence for a preventive effect of statins in cancer, including lymphoma. So far, nothing is known about a therapeutic activity of statins or related drugs in Hodgkin lymphoma. Statins block the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to downstream isoprenoid products like farnesyl and geranylgeranyl-pyrophosphate, inhibiting protein prenylation and leading to apoptosis in several malignancies. The anti-tumour activity of the statin simvastatin and three specific prenylation inhibitors (FTI-277, GGTI-298 and tipifarnib) in Hodgkin lymphoma cells was evaluated in cytotoxicity and apoptosis assays. Furthermore, simvastatin was tested in a xenograft model for human Hodgkin lymphoma. First simvastatin induced caspase-related apoptosis in Hodgkin lymphoma cells via the isoprenoid pathway. The IC50 of simvastatin in cytotoxicity assays was < 2 μM. Second simvastatin showed high activity in a mouse model for Hodgkin lymphoma, delaying tumour establishment and inhibiting tumour growth. Finally the geranylgeranyltransferase inhibitor GGTI-298 and the farnesyltransferase inhibitors FTI-277 and tipifarnib displayed anti-tumour activity in Hodgkin lymphoma cells (approximate IC50 in cytotoxicity assays: 20, 7.5 and < 0.1 μM respectively). Importantly, the concentration of simvastatin that induced apoptosis in Hodgkin lymphoma cells was in the range of 2 μM. This corresponds to plasma levels reached in humans in clinical trials with high dose statins in cancer. Thus, our results suggest two possible applications of simvastatin in Hodgkin lymphoma: First simvastatin in high doses could be included in polychemotherapy regimens to improve the remission rate of relapsed patients. Second simvastatin in the cholesterol-lowering dose should be evaluated as a preventive drug for patients in remission to avoid relapses. Moreover, in comparison to other haematological malignancies, the farnesyltransferase-inhibitor tipifarnib was highly cytotoxic in Hodgkin lymphoma cells. Given its activity in clinical trials in haematological neoplasia, its clinical evaluation in Hodgkin lymphoma is warranted. Taken together, prenylation inhibitors are promising drugs for the treatment of Hodgkin lymphoma.
Evidence for an Underestimation of the Prognostic Impact of Poor Cytogenetics within the IPSS.
Authors: Haase Detlef , Germing Ulrich , Schanz Julie , Pfeilstoecker Michael , Hildebrandt Barbara , Luebbert Michael , Giagounidis Aristoteles , Aul Carlo , Truemper Lorenz , Mueller Thomas , Valent Peter , Gattermann Norbert , Fonatsch Christa , Krieger Otto , Stauder Reinhard , Steidl Christian
Published: October 2019

In the IPSS the variables bone marrow blasts, cytogenetics and cytopenias were found to be most relevant for clinical outcome by multivariate analysis. Comparing cytogenetics and blasts scoring points were assigned as follows: 0 (good cytogenetics and less than 5% blasts), 0.5 points (intermediate cytogenetics, and 5–10% blasts), 1.0 points (poor cytogenetics), 1.5 points (11–20% blasts), 2.0 (21–30% blasts). In order to examine the correctness of weighting of cytogenetics in comparison to blast counts we compared the survival curves, median survival times (mst) and differences of mst (mst diff.) related to the mst of 37.5 months (mo) of our entire study population (on the basis of 2124 pts. with MDS from our German-Austrian database) between patient subgroups. The results in the subgroups were as follows: Blasts below 5% (n=609) (mst: 58 mo, mst diff.: +20.5 mo), good cytogenetics (n=768) (mst: 55.3 mo, mst diff.: +17.8 mo), blasts 5–10% (n=231) (mst: 28.0 mo, mst. diff.: −9.5 mo), intermediate cytogenetics (n=222) (mst: 28.0 mo, mst diff.: −9.5 mo), blasts: 11–20% (n=160) (mst: 16.5 mo, mst diff.: −21 mo), poor cytogenetics (n=212) (mst: 11.1 mo, mst diff.: −26.3 mo), blasts 21–30% (n=92) (mst: 11.7, mst diff.: −25.7 mo). Our results clearly show that within the IPSS poor cytogenetics are significantly underweighed. Referring to the survival data unfavorable cytogenetics resulted in a survival disadvantage at the same scale as compared to 21–30% blasts. Thus, in a revised IPSS this cytogenetic feature should get the same scoring points as compared to 21–30% blasts when using the FAB-classification. For a scoring system based on the WHO-classification unfavorable cytogenetics should get an even higher scoring value as compared to the maximum blast count of 19%. Further statistical analyses are on the way to substantiate our conclusions.
Targeting BH3-Domain Anti-Apoptotic Proteins with GX15-070 Decreases DNA Synthesis, Induces Cell Death and Sensitizes Rituximab-Sensitive and Resistant Non-Hodgkin’s Lymphoma Cell Lines to the Anti-Tumor Activity of Chemotherapy Agents.
Authors: Hernandez-Ilizaliturri Francisco J. , Iqbal Arshad , Alam Naheed , Bhat Seema , Olejniczak Scott , Knight Joy , Czuczman Myron S.
Published: October 2019

GX15-070 is a potent pan-bcl-2 inhibitor with known activity against chronic lymphocytic leukemia (CLL) cells and mantle cell lymphoma (MCL) cell lines, currently undergoing phase I testing. Bcl-2 over-expression is associated with chemotherapy resistance and correlates with a poor clinical outcome in diffuse large B-cell lymphoma. Recently, we demonstrated that acquisition of rituximab resistance is associated with deregulation of BH3-domain pro- and anti-apoptotic proteins. This leads to concomitant resistance to multiple chemotherapy agents. Targeting BH3-domain anti-apoptotic proteins with GX15-070 is an attractive strategy to potentially overcome acquired biologic and/or chemotherapy resistance. To this end we studied the effects of GX15-070 in a panel of rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL). Resistant clones were generated by chronic exposure of Raji, RL or DHL-4 cells to escalating doses of rituximab with (4RH) or without (2R) human complement. Functional assays (e.g. ADCC and CMC assays) demonstrated a significant decrease in rituximab sensitivity in RRCL. In addition, resistance to chemotherapy agents (CDDP, doxorubicin, vincristine, etc.) was demonstrated in RRCL. Lymphoma cells were exposed in vitro to GX15-070 (0 to 20mM) with or without CDDP (0 to 10mM) or Doxorubicin (0 to 1mM). Following a 24 and a 48 hour period of drug exposure: induction of apoptosis and the percentage of viability was determined by flow cytometric analysis, Western blotting and trypan-blue staining techniques. In addition, changes in DNA synthesis and cell proliferation following drug exposure were performed using standard [3H]-thymidine incorporation assays. GX15-070 induced a dose-dependent cell death and decrease in DNA synthesis in all the cell lines tested (both RSCL and RRCL). Up to 75% of cell death was observed in all cell lines exposed to 20mM of GX15-070. Anti-tumor activity was seen even at the lowest tested dose of GX15-070 (2mM, 20–25% death cells). Incubation of RSCL and RRCL with 2 or 5mM of GX15-070 induced synergistic cytotoxic and anti-proliferative effects when combined with CDDP and doxorubicin. The IC50 of CDPP and doxorubicin was decrease ten-fold by the concomitant in vitro exposure to GX15-070 in all cell lines tested, including RRCL. Our data demonstrates that GX15-070 is active against both RSCL and RRCL and augments the anti-tumor activity of chemotherapy agents. These results strongly suggest that GX15-070 added to systemic chemotherapy is a potentially valuable and novel therapeutic strategy in the treatment of B-cell NHL.
Monocyte Colony-Stimulating Factor Enhances Rituximab-Dependent Cellular Cytotoxicity by Monocytes.
Authors: Takami Akiyoshi , Shimadoi Shigeru , Kondo Yukio , Okumura Hirokazu , Nakao Shinji
Published: October 2019

[Introduction]

Recent data suggest that monocytes are the dominant effector cells during immunotherapy using the anti-CD20 monoclonal antibody rituximab, depleting B cells through FcγR-dependent pathways. Because monocyte colony-stimulating factor (M-CSF) enhances the antibody-dependent cellular cytotoxicity (ADCC) of monocytes, the clinical efficacy of rituximab might be improved by the addition of M-CSF. We have studied the effect of M-CSF in the enhancement of rituximab-mediated ADCC against B-cell lymphoma.

[Methods]

Monocytes were isolated by negative selection of PBMCs from healthy individuals for the absence of T-cell, B-cell, and NK-cell markers. Cytotoxicity was determined by a flow cytometry using two fluorescent dyes, calcein-AM and ethidium homodimer to specifically stain living and dead cells respectively. Monocytes were cultured for 48 hours in the presence or absence of human recombinant M-CSF (66 ng/ml). The B-cell lymphoma cell line Daudi was used as target in the presence of rituximab (5 μg/ml) or human IgG1 as control for 30 min at room temperature. Effector cells and target cells were incubated at different ratios ranging from 1:1 to 15:1 for 4 hours at 37°C. The expression of FcγRl, FcγRII, and FcγRIII on monocytes was determined using a flow cytometry.

[Results]

Monocytes treated with M-CSF showed a significant increase in rituximab-mediated cytotoxicity against B lymphoma cells: specific lysis at an E:T ratio of 15:1 was 39% ± 7% (mean ± SD) vs. 21% ± 5%, M-CSF-treated monocytes vs. non-treated monocytes. Lysis of lymphoma cells treated with rituximab alone was 8% ± 4%. Treatment with M-CSF led to a 1.5- to 2.0-fold increase of FcγRI and FcγRIII expression in monocytes, while FcγRII expression remained unchanged.

[Conclusion]

Pretreatment of monocytes with M-CSF enhances their rituximab-mediated ADCC against B-cell lymphoma, which may partly result from increasing expression of FcγRI and FcγRIII on monocytes via M-CSF stimulation. These in vitro results may provide a new approach to improve the therapeutic activity of rituximab.
Osteosarcoma Derived from Cultured Mesenchymal Stem Cells.
Authors: Tolar Jakub , Nauta Alma J. , Osborn Mark J. , Panoskaltsis-Mortari Angela , McElmurry Ron T. , Bell Scott , Xia Lily , Zhou Ning , Riddle Megan , Schroeder Tania M. , Westendorf Jennifer J. , McIvor R. Scott , Hogendoom Pancras C.W. , Szuhai Karoly , Oseth LeAnn , Hirsch Betsy , Yant Stephen R. , Kay Mark A. , Peister Alexandra , Prockop Darwin J. , Fibbe Willem E. , Blazar Bruce R.
Published: October 2019

The beneficial effects of Mesenchymal Stem Cells (MSC) are being tested clinically in attempts to improve hematopoietic engraftment, to treat osteogenesis imperfecta, graft-versus-host disease and autoimmune diseases, and to deliver therapy for malignancies. In early reports, phase I clinical studies have not been associated with toxicities. To study the biodistribution of MSC, we labeled adult murine C57BL/6 MSC with firefly luciferase and DsRed2 fluorescent protein using non-viral Sleeping Beauty transposons, and co-infused labeled MSC with bone marrow into irradiated allogeneic recipients. Using in vivo whole body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in lungs. Two mice also developed sarcomas in their extremities. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Mapping of the Sleeping Beauty transposition insertion sites did not identify an obvious transposon-related genetic abnormality. Importantly, the original MSC cultures not labeled with transposons, as well as cultured MSC independently isolated from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro. Even though not all MSC cultures formed tumors upon in vivo injection, these data indicate that MSC transformation was neither strain-specific nor a rare event following ex-vivo expansion. Karyotype analyses using fluorescence in situ hybridization with spectral karyotyping (SKY) as well as combined binary ratio labeling of nucleic acid probes (COBRA) showed clonal evolution of transformed MSC suggesting that the critical transformation event(s) occurred before MSC infusion. Collectively, we describe cytogenetic instability of murine MSC isolated in two independent laboratories, their cellular transformation, and potential for sarcoma formation. While the growth characteristics of human and murine MSC are not identical and murine cells are more prone to undergo immortalization and transformation in culture than human cells, our study highlights the importance of quality control measures needed for ongoing and future clinical trials using human MSC.
Optimizing Mobilization, Augmentation, and Gene Transfer of Multipotent Mesenchymal Stromal Cells (MSCs).
Authors: Larsen Stephen R. , Chng Keefe , Zhou Shangzhen , Wright John , Armstrong Margaret , Thompson Sally , Hennessy Annemarie , Martiniello-Wilks Rosetta , Gibson John , Joshua Douglas E. , Rasko John E.J.
Published: October 2019

Aims: MSCs are cells being investigated for use in various therapies including facilitation of HSC transplantation and as gene therapy delivery vehicles. We have explored the potential to increase the number of bone marrow (BM) MSCs in vivo, induce mobilization using various cytokine regimens and improve gene transfer into these cells with adeno-associated virus (AAV) in a baboon model.

Method: Baboons received cytokines as follows: 1. G-CSF 100mcg/kg/day for 5 days; 2. pegylated G-CSF (pegG-CSF), single dose 300mcg/kg day −5; 3. G-CSF 100mcg/kg/day + stem cell factor (SCF) 50mcg/kg/day for 5 days; and 4. pegylated megakaryocyte growth and development factor (pegMGDF) 1mcg/kg second daily for 10 days + G-CSF 100mcg/kg/day for 5 days starting day −5. Animals underwent BM aspiration at baseline and on the final day of cytokines along with leukapheresis to isolate PBMNCs for detection of peripheral blood (PB) CFU-F. The immunophenotype and differentiation potential of CFU-F derived from animals before and after cytokines was compared. The ability of AAV vectors pseudotyped with capsids derived from AAV of serotypes 1, 2, 3, 4, 5, 6, and 8 to mediate transduction of baboon and human MSCs was assessed.

Results: Augmentation of bone marrow MSCs was observed with all cytokine regimens with the fold-increase compared to baseline as follows: 4.1, 2.1, 7.6 and 11.2 after G-CSF, pegG-CSF, G-CSF+SCF and G-CSF+pegMGDF respectively (see Figure 1). The immunophenotype of MSCs obtained after cytokines was identical to baseline cells as was their differentation potential. CFU-F were not detected in baseline PB however they were detected in 3/5 animals after G-CSF+SCF at a frequency of 0.8 to 1.5/mL, but no other cytokine regimen. A similar pattern of transduction efficiency using AAV was shared by human and baboon MSCs (see Figure 2) using control Ad293 cells. Specifically AAV vectors expressing capsids of serotypes 2, 3 and 5 were most efficient in transducing human and baboon MSCs. Those expressing capsids from serotypes 1, 4, 6, and 8 were much less efficient in transducing MSCs from either species. Baboon MSCs were able to be transduced by about 100-fold more than their human equivalent cells using AAV serotypes 2, 3, and 5.

Conclusion: This is the first report of mobilization of primate MSCs and, together with the demonstration of in vivo augmentation and AAV gene transfer, offers increased therapeutic opportunities for their safe application in a burgeoning number of diseases.

Figure 1: In vivo Bone Marrow CFU-F Augmentation following cytokines Figure 1:. In vivo Bone Marrow CFU-F Augmentation following cytokines Figure 2: Transduction profile of AAV vectors expressing capsids of various serotypes Figure 2:. Transduction profile of AAV vectors expressing capsids of various serotypes
The Novel HDAC Inhibitor UCL67022 Is Highly Potent in Multiple Myeloma and Non-Hodgkin’s Lymphoma and Is Enhanced by Bortezomib.
Authors: Maharaj Lenushka , Popat Rakesh , Chahwan Angela , Lister Andrew T. , Cavenagh James D. , Middleton Brian , Rioja Alf , Marson Charles , Joel Simon P.
Published: October 2019

Introduction: Histone deacetylases (HDACs) regulate the acetylation state of nucleosomal histones and represent a novel target in haematological malignancies. Inhibition of HDACs results in the induction of apoptosis in multiple myeloma (MM) and lymphoma (NHL) cells, associated with the down-regulation of signaling pathways involving IL-6 and IGF-1 and the inactivation of BCL-6. We have therefore investigated the activity of a novel hydroxamic acid HDAC inhibitor (UCL67022) in comparsion with SAHA, alone and in combination with the proteasome inhibitor bortezomib.

Methods: HDAC activity in partially purified rat liver homogenate and intact CEM cells was determined using a fluorescent HDAC substrate. The human MM cell lines RPMI 8226/S and U266, and the NHL cell lines SUD-4, CRL, DOHH2, DHL-4, 5, 6, 7, GRANTA-519 and JEKO-1 were used to investigate effects on viable cell number using an ATP-dependent bioluminescence method. Activity against primary malignant cells from patients with MM, DLBCL, FL or CLL was also studied. Western blot analysis was used to investigate changes in acetylated histone-H3 and a-tubulin.

Results: UCL67022 showed more potent HDAC inhibitory activity in liver extracts and whole CEM cells than SAHA (IC50 0.05 vs 0.39uM in liver and 0.11 vs 0.33uM in CEM cells). UCL67022 was more potent than SAHA in reducing viable cell number in U266 (EC50 0.14 vs 1.8 uM) and RPMI 8226/S (0.05 vs 0.78 uM) cells. Similarly NHL cell lines were 10–20 fold more sensitive to UCL67022, with median EC50 values of 0.05uM (range 0.03–0.10uM) vs 0.81uM, (range 0.68–1.28uM). In 2 primary MM samples UCL67022 showed increased activity with EC50 values of 0.7uM and 0.11uM vs 12.2uM and 1.1uM for SAHA. 3 FL patient samples and 1 CLL sample were 3.7-fold more sensitive to UCL67022 than SAHA (median EC50 4.7uM vs 17.5uM respectively). Western blot analysis showed a 10-fold difference in histone H3 acetylation between the two compounds, with acetylation returning to pre-treatment levels by 24hr with 3uM SAHA but remaining elevated out to 48hr with 0.3uM UCL67022. Increased acetylation of a-tubulin confirmed the inhibition of HDAC6. Combination with bortezomib at 1, 2 and 4nM in MM cells showed increased antiproliferative activity with both SAHA and UCL67022, with synergistic responses observed in the U226 cell line and in 1 primary sample and additive effects in the others.

Conclusions: These data demonstrate the activity of the highly potent, novel HDAC inhibitor UCL67022 in MM and NHL cell lines and primary patient cells. Activity was increased in co-exposures with bortezomib possibly due to inhibition of HDAC6 and subsequent aggresome formation, suggesting a therapeutic advantage with the combination.
FLT-PET in Haematological Disorders: A Novel Technique To Analyze the Proliferative Activity of the Bone Marrow Compartment.
Authors: Vellenga Edo , Agool Ali , Schot Bart , Jager Piet
Published: October 2019

Few diagnostic procedures are available to determine the degree of bone marrow cellularity and the numbers of cycling cells in patients with hematopoietic disorders. Therefore non-invasive imaging of the bone marrow compartment may be helpful. Recently the PET tracer [18F]-3′-fluoro-3′-deoxy-L-thymidine (FLT) has been developed. FLT uptake is related to the DNA synthesis rate and increases with higher proliferation rates and may therefore visualise the high cycling activity of haematopoietic cells in the bone marrow compartment. In the present study the feasibility of visualisation and quantification of the bone marrow compartment activity with FLT-PET was used to distinguish different haematological disorders.

Clinical and laboratory data of 18 patients with low-risk myelodysplasia (MDS), chronic myeloproliferative disorders, myelofibrosis, aplastic anemia or multiple myeloma were correlated with the results of FLT-PET imaging using visual analysis and SUV. Findings were compared with those in normal controls (n=14).

With SUV analysis of the femura, thoracic and lumbar vertebral body in conjunction with visual analysis a distinction could be made between MDS (n=9), chronic myeloproliferative disorders (n=3) and myelofibrosis (n=3) compared to normal controls (p<0.001). A significant increase in FLT uptake in the defined areas was observed in all the studied patients with MDS and myeloproliferative disorders. In addition a significant expansion of the bone marrow compartment was shown (p<0.001). No correlation was found between Ki-67 staining of bone marrow biopsies and FLT uptake. In contrast, patients with myelofibrosis demonstrated a decline in bone marrow FLT uptake compared to normal controls but a significant increase in liver and spleen uptake. Osteolytic lesions of multiple myeloma patients and patients with aplastic anemia showed a strong reduction in bone marrow FLT uptake.

The results indicate that FLT-PET can be used to visualize the proliferative activity of the bone marrow compartment and may be helpful to distinguish separate haematological disorders.
Hepcidin Expression Is Regulated by a Complex of Hemochromatosis-Associated Proteins.
Authors: Schmidt Paul J. , Huang Franklin W. , Wrighting Diedra M. , Toran Paul T. , Andrews Nancy C.
Published: October 2019

Hemochromatosis is a common genetic disease resulting from increased dietary iron absorption and tissue iron deposition. Mutations in five unrelated genes are known to cause hemochromatosis in humans and mice. These encode the classic hemochromatosis protein (HFE), transferrin receptor 2 (TFR2), the iron exporter ferroportin (FPN), hemojuvelin (HJV), and the circulating anti-microbial peptide hepcidin (HAMP). Hepcidin binds to FPN, causing its internalization and degradation, thus decreasing cellular iron release. A basic understanding of the pathophysiology of FPN and hepcidin mutations has recently been elucidated; however, it was still unclear how mutations in HFE, TFR2, and HJV cause hemochromatosis. All are associated with decreased hepcidin production and inappropriately high levels of ferroportin activity. HFE, TFR2 and HJV are normally expressed in the hepatic cells that produce hepcidin. With collaborators, we showed that HJV acts as a bone morphogenetic protein (BMP) co-receptor. HJV binds to the BMP ligands and forms a complex with Type I BMP receptors, resulting in signaling through a SMAD pathway and induction of hepcidin expression. Disease causing mutations in HJV abrogate BMP co-receptor activity, and hepatocytes from Hjv−/ − mice have a blunted response to BMP2. HFE was known to form a complex with the classical transferrin receptor, TFR1. Several models have been proposed implicating this complex in the regulation of normal iron homeostasis, but they have not taken the role of hepcidin into account. To examine the HFE/TFR1 interaction in vivo, we developed mice expressing a mutant form of TFR1 that should constitutively interact with HFE. We found that these transgenic animals have a phenotype similar to Hfe−/ − mice, suggesting that TFR1 serves to sequester HFE to silence its activity. We next asked whether HFE might also participate in BMP signaling. We found that forced expression of HFE in a hepatoma cell line induces transcription of a reporter gene linked to the hepcidin promoter. It also induces transcription from a heterologous promoter containing BMP-responsive elements, suggesting that HFE works through the BMP pathway. In contrast, forced expression of TFR2 did not amplify expression of either reporter, but it prevented cellular release of a soluble cleavage product of HJV. Furthermore, we showed that both HFE and TFR2 are associated with HJV in a stable protein complex that can be isolated by co-immunoprecipitation or Ni-affinity chromatography. TFR2 appears to aid in the recruitment of HFE to this complex. We conclude that HFE and TFR2 thus serve to amplify BMP signaling through an HJV/BMP receptor pathway. Our findings provide a compelling explanation for the similar clinical hemochromatosis phenotypes resulting from mutations in these genes.
Phase II Results of Bendamustine in Combination with Rituximab in Relapsed Indolent and Mantle-Cell Non-Hodgkin’s Lymphoma.
Authors: Van der Jagt Richard H. , Cohen Philip , Cheson Bruce D. , Tulpule Anil , Herst Jordan A. , Lemieux Bernard , Schwartzberg Lee S. , Robinson Katherine Sue , Oliver Jennifer W. , Williams Michael E.
Published: October 2019

Objective: The objective of this study was to evaluate the efficacy and safety of bendamustine HCl (TREANDA®) in combination with rituximab in patients with relapsed non-Hodgkin’s lymphoma (NHL).

Background: Bendamustine is a novel hybrid, alkylating agent with single-agent activity in multiple hematologic and solid tumors. It induces cell death via both apoptosis and the apoptosis-independent pathway of mitotic catastrophe. The combination of bendamustine and rituximab has been shown to exhibit a synergistic antitumor effect on NHL cells.

Methods: This Phase II, multicenter study enrolled adult patients with relapsed, indolent B-cell or mantle-cell NHL who were not refractory to rituximab (defined as progression ≤6 months of last rituximab dose). Patients received rituximab 375 mg/m2 intravenously (IV) on day 1 and bendamustine 90 mg/m2 IV on days 2 and 3 of a 28-day cycle for 4 to 6 cycles. An additional dose of rituximab 375 mg/m2 IV was given 1 week before the first cycle of bendamustine and 4 weeks after the last cycle.

Results: The intent-to-treat (ITT) population included 66 patients (59% men) with a median age of 60 years (range, 40–84). Indolent histologic phenotype was seen in 54 patients with the following histologic subtypes: follicular center cell (61%), small lymphocytic (15%), lymphoplasmacytic (3%), and marginal zone (3%); 18% had mantle-cell lymphoma (MCL). A total of 85% of patients had stage III/IV disease. These patients relapsed from a median of 1 prior chemotherapy (range: 0–5), with 56% having had prior treatment with rituximab. Patients with no prior chemotherapy relapsed following biologic therapy. In the ITT population, the overall objective response rate (ORR) was 94% (complete response [CR]/complete response unconfirmed [CRu], 41%; partial response [PR], 53%); 6% had stable disease. The ORR for the 12 MCL patients was 92% (CR/CRu, 42%; PR 50%). For all patients, the median duration of response and progression-free survival has not been reached after a median follow-up of 8.3 months (range, 0.14–31 months). Grade 3/4 neutropenia was seen in 41% of patients (7%, febrile neutropenia). Common nonhematologic toxicities (grade 1/2, grade 3, grade 4) were nausea (68%, 0%, 0%) and fatigue (53%, 5%, 0%); one patient had grade 3 sepsis. No alopecia was observed.

Conclusions: Bendamustine administered in combination with rituximab produced a high objective response rate and was generally well tolerated in patients with relapsed indolent and mantle-cell NHL who were not refractory to rituximab. These results suggest that the combination of bendamustine and rituximab may be comparable in activity to R-CHOP, and further studies of this combination are warranted.
Combination Immunotherapy with a CpG Oligonucleotide and Rituximab (R) Augments the Immunological Response and Favorably Alters the Malignant Microenvironment of Follicular Lymphoma (FL).
Authors: Friedberg Jonathan W. , Kelly Jennifer , Kutok Jeffrey L. , Salloum Rabih , Fisher David C. , Ronan Elizabeth , Dalton Virginia , Rich Lynn , Marquis Diana , Rothberg Paul G. , Liesveld Jane , Fisher Richard I. , Coffman Robert , Mosmann Tim , Freedman Arnold S.
Published: October 2019

The malignant microenvironment of FL, including T cells, macrophages and follicular dendritic cells, contributes to disease pathogenesis and represents a rational therapeutic target. Gene expression profiling and immunohistochemistry studies have confirmed that degree and type of immune response in the malignant FL lymph node impacts clinical outcome. CpG oligonucleotides have pleotropic effects on both the innate and adaptive immune systems, including increased antigen expression, enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and shifting the immune response in a TH-1 direction. These agents therefore have significant potential to manipulate the immune response to FL, and alter the malignant microenvironment in a favorable way. We combined a CpG oligonucleotide (1018 ISS, 0.2 mg/kg sc weekly x 4 beginning day 8) with standard R in patients (pts) with relapsed/refractory, histologically confirmed FL, and comprehensively evaluated immunological changes following the combination. 22 pts (10 female; median age 55) with FL sensitive or naive to R were enrolled. FcγRIIIA polymorphism status was FF n=9, VF n=12, VV n=1. Treatment was well-tolerated with no significant adverse events attributable to therapy. Baseline and post therapy evaluations were performed on blood and the CpG injection sites in all pts. Compared to baseline, 7 pts demonstrated significant increases in ADCC after therapy using a conventional chromium-release assay. Circulating CD3-positive cells increased in 11 pts. Increased IFN-γrelease by Elispot occurred in 5 pts. 10 pts had significant increases in neutrophils, T cells, and macrophages in skin biopsies of CpG injection sites performed 24 hours post therapy compared with baseline. Pre- and post-biopsies of tumor tissue (including lymph nodes and bone marrow) were performed in 5 pts, and in all cases a significant infiltration of T cells (CD8 > CD4) and macrophages were observed following treatment. Clinical responses per Cheson criteria were observed in 45% of pts; median PFS was 18 months in responding pts, and 9 months for entire group; 12 pts have not yet required further therapy for FL. In conclusion, this group of pts with relapsed FL had favorable clinical outcome despite adverse prognostic factors. The addition of CpG to R had measurable and reproducible local and systemic immune effects in a substantial subset of pts. Unlike other cytokine/antibody combinations, this study is the first to histologically confirm perturbation of the local immune malignant microenvironment following systemic biological therapy of FL.
Prognostic Significance of Quantitative t(14;18) PCR Monitoring in Advanced Stage Follicular Lymphoma Patients.
Authors: Hirt Carsten , Schueler Frank , Kiefer Thomas , Haas Antje , Niederwieser Dietger , Neser Sabine , Assmann Michael , Dachselt Klaus , Leithaeuser Malte , Rabkin Charles S. , Herold Michael , Dolken Gottfried
Published: October 2019

In a prospective multicenter phase III trial (M 39023) advanced stage follicular lymphoma (FL) patients were randomized to receive eight cycles of MCP (mitoxantrone, chlorambucil, prednisolone) chemotherapy alone or in combination with rituximab (R-MCP). This study was carried out to determine the effects of this therapy on circulating lymphoma cells (CLC) and to assess the value of CLC quantitation as a molecular marker of disease activity and as a prognostic parameter. CLC numbers were determined by real-time quantitative PCR for the t(14;18)-MBR translocation or by allele-specific PCR for rearranged immunoglobulin heavy chain genes. Quantitative PCR of a reference gene (wild type K-ras) allowed the exact quantitation of cells tested per sample and to exclude samples with insufficient DNA content (< 500,000 cells). We analyzed serial blood samples from 43 patients obtained before, during and after completion of therapy. Baseline clinical characteristics and response to therapy of the 43 patients of this study were not significantly different to all 201 FL patients of the clinical trial except for a higher complete response (CR) rate in the R-MCP group (18/25 (72%) in this study versus 52/105 (50%) in the clinical trial, p=.04). Similar to the results of the clinical study, response rate and CR rate in the present study were significantly higher in the R-MCP arm than after therapy with MCP alone (25/25 (100%) versus 13/18 (72%), p= .009, and 18/25 (72%) versus 5/18 (28%), p= .006, respectively). Clearance of CLC at the end of therapy was achieved in 21/25 patients (84%) treated with R-MCP compared to 0/18 (0%) after MCP alone (p< .0001). R-MCP patients achieved a greater reduction of CLC after completion of therapy and a greater reduction of CLC per treatment cycle than patients treated with MCP alone, even if the comparison was restricted to patients with clinical response (3.88 log and 1.18 log reduction versus 2.21 log and 0.23 log reduction, p= .001 and p< .0001). A ≥ 2 log reduction of CLC after completion of therapy was associated with a favourable clinical response (p= 0.0007) and prolonged event-free survival (p= 0.02) regardless of treatment arm. Among patients with a ≥2 log CLC reduction there was no significant difference in EFS between patients with PCR negative samples and patients with PCR positive blood samples at completion of therapy (p= 0.091). In conclusion, R-MCP led to a rapid and sustained eradication of CLC in the majority of patients. The results of serial determinations of CLC numbers showed a good correlation with the quality and duration of the clinical response. Therefore, quantitative molecular disease monitoring could help to develop individualized treatment strategies for patients with advanced stage FL.
Efficacy of 90Y-Ibritumomab Tiuxetan (Zevalin) in Refractory or Relapsed MALT Gastric Non Hodgkin’s Lymphoma.
Authors: Vanazzi Anna , Ferrucci Pier F. , Grana Chiara , Pruneri Giancarlo , Crosta Cristiano , Pinto Antonello , Chinol Marco , Paganelli Giovanni , Martinelli Giovanni
Published: October 2019

Radio-immunotherapy (RIT) is an effective approach for patients with relapse-refractory NHL. The aim of RIT is to use the monoclonal antibody to target radiation to the tumor bulk while limiting exposure of normal tissues. Toxicity is prevalently hematologic, but transient and reversible. Zevalin in particular has demonstrated activity in follicular (FL) and diffuse large B-cell (DLBC) NHL, but data are still lacking in MALT NHL. In this setting of pts Zevalin could offer an alternative option to those pts who relapse or do not respond to conventional treatment. We report efficacy results of a pilot study of Zevalin delivered at conventional activity (0,4 mCi/kg) in gastric NHL pts resistant or refractory to conventional systemic treatment. From May 2004 to January 2006, 7 patients were enrolled. They all had gastric resistant-refractory CD-20 positive B-cell NHL: 2 DLBC and 5 MALT. Median age was 57 ys (range 36–64), 3 female and 4 male. At time of treatment 5 out of 7 patients had stage I/IIA (MALT), while 2 out of 7 pts had stage III/IV (DLBCL) of disease. Helicobacter Pylori was negative in all pts at time of treatment, positive at diagnosis in 3 out of 5 pts affected by MALT gastric NHL. Median number of previous therapies received was 2 (1–4): all of them had received prior CT, 3 prior Rituximab, no one had received prior RT. Toxicities were primarily haematological, as expected, and reversible. Response evaluation was performed two months after Zevalin administration by upper gastrointestinal endoscopy with multiple biopsy and endoscopic ultrasonography. All patients are now evaluable for response: 5 CR (all MALT), 2 PD (both DLBC NHL). All responders still maintain a CR at 27, 20, 16, 9 and 8 months after treatment.

Conclusions: Zevalin seems to be very active in pts with MALT gastric NHL resistant or refractory to conventional systemic treatment. Its mechanism of action mimics conventional RT already known as valid option in MALT gastric NHL. If these preliminary results are confirmed in larger number of pts, one single administration of Zevalin delivered at conventional activity (0,4 mCi/kg) could be considered a possible alternative option in the treatment of such indolent disease.
Smudge Cells on Routine Blood Smear Predict Clinical Outcome in Chronic Lymphocytic Leukemia: A Universally Available Prognostic Test.
Authors: Nowakowski Grzegorz S. , Hoyer James D. , Shanafelt Tait D. , Geyer Susan M. , LaPlant Betsy , Call Timothy G. , Jelinek Diane F. , Zent Clive S. , Kay Neil E.
Published: October 2019

Background: The prognosis of patients with early Rai and Binet stage CLL is variable. Recently developed prognostic tests (mutation status, ZAP-70, chromosome evaluation by FISH) require significant technologic expertise and are not available worldwide to most patients with CLL. Smudge cells are ruptured CLL cells appearing on a blood smear. Using a proteomic approach, we recently discovered that the cytoskeletal protein vimentin is expressed at higher levels in CLL cells with unmutated IgVH and that vimentin expression is a marker of poor prognosis in early stage CLL (Blood, 2005;106:707).

Hypothesis: Since vimentin plays an important role in the cytoskeleton and lymphocyte rigidity, we hypothesized that smudge cell formation is inversely correlated with vimentin expression and that the percentage of smudge cells may predict prognosis in CLL.

Methods: We blindly reviewed the blood smears archived at the time of diagnosis of patients with untreated, early stage CLL. A total of 200 lymphocytes and smudge cells were counted per slide and results reported as percentage of total lymphocytes (intact and smudged). The percentage of smudge cells was correlated with vimentin expression measured by flow cytometry, IgVH mutation status, time to treatment (TTT), and survival.

Results: Blood smears of 75 patients were reviewed. The median percentage of smudge cells was 27% (range, 4–72%). The percentage of smudge cells did not correlate with lymphocytosis (r=0.04, p=0.73) and did not change overtime for serial evaluations for individual patients (p=0.61). The percentage of smudge cells appeared to correlate inversely with vimentin expression (r = − 0.627; p = 0.0017). The percentage of smudge cells was higher in patients with mutated IgVH than in patients with unmutated IgVH (median 31% vs. median 13%, p=0.02). The percentage of smudge cells as a continuous variable correlated with both a prolonged TTT (p=0.0023) and overall survival (p=0.04). Patients with less than 30% smudge cells had a median TTT of 72.7 months. The median TTT in patients with ≥30% smudge cells was not reached (p=0.0011), Figure 1.

Conclusion: A lower percentage of smudge cells is associated with unmutated IgVH status and with worse clinical outcome in early stage CLL. The percentage of smudge cells does not vary based on the magnitude of the lymphocytosis, but is patient specific and remains constant over time. Evaluation of the percentage of smudge cells on blood smears could be a universally available prognostic test for patients with early stage CLL.

Figure Figure
Highly Purified Hematopoietic Stem Cell Can Generate Cardiomyocytes Via Myeloid Progenitors through Cell Fusion.
Authors: Fukata Mitsuhiro , Ishikawa Fumihiko , Shimazu Hideki , Nakamura Ryu , Shimoda Kazuya , Akashi Koichi , Leonard Shultz D. , Harada Mine
Published: October 2019

[Introduction] Recent reports demonstrating the ability of bone marrow (BM) cells to regenerate cardiomyocytes have prompted clinical as well as basic studies for the treatment of ischemic and non-ischemic cardiomyopathies. However, the responsible BM cells and underlying mechanism of regeneration remain unclear. In this study, we checked the cardiomyogenic potential of BM cells in vivo, to identify the cell populations in BM that possess the capacity to give rise to cardiomyocytes and clarify whether cardiomyogenic potential of BM-derived cells require cell fusion with host cardiomyocytes.

[Method and Result] BM cells of mice constitutively expressing green fluorescent protein (GFP) was intravenously injected into irradiated syngeneic newborn C57BL/6 mouse within 48hours after the birth. At 4–5 weeks after transplantation, the cardiac tissue of recipients was analyzed for the number of GFP+ cardiomyocytes per 40 sections. We first separated Linaege antigen− BM cells to CD45+ cells (Lin−CD45+) and CD45− cells (Lin−CD45−) and transplanted each. In the recipients of Lin−CD45+, 37.0±23.9 GFP+ cardiomyocytes were detected (n=6). While no GFP+ cardiomyocyte was detected in the recipients of Lin−CD45− (n=4). Since hematopoietic cells generated cardiomyocytes, we next transplanted limiting numbers of purified HSCs and hematopoietic progenitors by cell sorting. Both HSCs and hematopoietic progenitors generated cardiomyocytes dose-dependently, suggested that HSC can generate cardiomyocytes via hematopoietic progenitors. To explore the hematopoietic lineage, we further transplanted purified myeloid progenitors and common lymphoid progenitors by cell sorting. The number of GFP+ cardiomyocytes were 12.8±10.7 and 0.91±1.20 in the recipients of myeloid progenitors (n=7) and common lymphoid progenitors (n=10) respectively (p<0.005), suggested that myeloid lineage is more strongly involved in the generation of cardiomyocytes than lymphoid lineage. Furthermore, we confirmed the contribution of myeloid cells to cardiomyocytes by transplanting common myeloid progenitors (CMPs) and granulocyte/macrophage lineage-restricted progenitors (GMPs). Finally, we transplanted GFP+ HSCs and myeloid progenitors into CFP (cyan fluorescent protein) transgenic mouse. Linear unmixing analysis using confocal microscopy revealed that donor-derived GFP+ cardiomyocytes coexpressed CFP, indicated cell fusion had occurred. We are now analyzing recipients transplanted with single GFP+ HSC.

[Conclusion] Our results suggested that HSC can contribute to the post-natal generation of cardiomyocytes via myeloid progenitors through cell fusion.