bluuebunny/crossref_metadata_2025 huggingface dataset:
Low-Dose Recombinante Urate Oxidase (Rasburicase) Is Effective in Hyperuricemia.
Authors: Trifilio S. , Tallman M. , Singhal S. , Gordon L. , Evens A. , Mehta J.
Published: October 2019

Hyperuricemia is a feature of tumor lysis syndrome (TLS), and is treated with hydration, urine alkalinization, and allopurinol. Allopurinol inhibits the conversion of hypoxanthine to xanthine, and xanthine to uric acid (UA) by inhibiting xanthine oxidase. It has no direct effect on existing UA. Rasburicase lowers UA rapidly to very low levels at the labeled dose of 0.15–0.2 mg/kg daily for 5 days by converting UA to allantoin which is rapidly excreted. Despite this dramatic effect on UA, rasburicase has not been shown to have any beneficial impact on survival. We administered 0.2 mg/kg ideal body weight rasburicase to an obese hyperuricemic patient (actual 120 kg, ideal 60 kg). Serum UA declined from 11 mg/dL to 1.4 mg/dL in 3 h and 0.4 mg/dL in 11 h. The serum UA level remained low for several days and a second dose was not needed. After seeing this dramatic and sustained response, low rasburicase doses were used in patients with malignant diseases with close monitoring of biochemical parameters. No dose level was systematically explored and the doses used ranged from 1.5 mg to 12 mg. This analysis is a retrospective review of all patients who received low-dose rasburicase. 23 adults (39–78 y) with cancer and hyperuricemia received a single low dose of rasburicase at 12 mg (n=1; index case), 6 mg (n=2), 4.5 mg (n=1), 3 mg (n=18), and 1.5 mg (n=1). 6 patients received 1 and 1 patient received 2 more doses of 3 mg each. Allopurinol and other supportive therapy including hydration were administered. UA levels (baseline 7.4–19 mg/dL, median 11.6) declined by 4–96% (median 40%) within 24 h of rasburicase administration (Fig 1; the dashed lines show patients receiving more than 1 rasburicase dose).

Figure Figure

The decline was 31–68% (median 46%) amongst the 12 patients receiving a single 3 mg dose (Fig 2).

Figure Figure

The baseline serum creatinine was 0.9–8.4 mg/dL (median 2.4), and the minimum and maximum values in the week following rasburicase were 0.7–5.8 (median 1.8) and 1.0–8.8 (median 2.6) respectively. No clinically significant renal dysfunction developed in any patient. The total rasburicase dose administered, 3–12 mg (median 3 mg), was 3–12% (median 5%) of the recommended dose. Based on the Red Book rasburicase cost of $387 for a 1.5 mg vial, the amount of money saved ranged from $11,000 to $29,000 per patient; for a total saving of between $373,000 and $507,000. While this was not a systematic dose-reduction study and patients were treated by multiple physicians and pharmacists on clinical grounds, the data suggest that rasburicase is effective at a fraction of the recommended dose. While a formal investigation of low doses is warranted, we recommend using rasburicase at a standard dose of 3 mg in hyperuricemic patients, checking UA levels frequently, and repeating rasburicase administration if needed. This approach has the potential for substantial cost saving while providing appropriate therapy to lower UA.
Respect of Average Relative Dose Intensity Correlates with Survival in Diffuse Large B Cell Lymphoma Patients Treated by CHOP: A National Retrospective Study (1995–2000).
Authors: Bosly Andre , Dominique Bron , Achiel Van Hoof , Robbrecht De Bock , Zwi Berneman , Augustin Ferrant , Gregor Verhoef
Published: October 2019

Aims: To describe the different regimens of chemotherapy (CT), the average relative dose (ARD) and dose-intensity (ARDI) for first line treatment of patients (pts) with diffuse large B-cell lymphoma (DLBCL).

Methods: 373 adult patients files from 29 centers receiving at least 3 cycles of treatment for DLBCL, included or not in a protocol between 1995 and 2000, were retrospectively reviewed. ARD and ARDI analysis (delivered vs planned) was only performed for cyclophosphamide (C) and anthracyclines (A) as they are considered to be the most important agents.

Results: 348 pts treated with CHOP-like, ACVBP-like or CHVmP-BV were evaluable for the analysis. CHOP-like was the most commonly used regimen (273 pts, 79 %) of which CHOP-21 (210 pts) was the most frequent; followed by ACVBP (57 pts, 16 %) (with ACVBP-14 (46 pts) as the most important type) and CHVmP-BV (18 pts, 5 %). For all regimens the relative total dose (RTD) effectively received was 96 % for C and 94 % for A, respectively, and the relative total dose intensity (RTDI) scored 92 % for C and 90 % for A. This results in an ARD of 94 % (SD 14 %) for CHOP, 99 % (SD 4 %) for ACVBP and 95 % (SD 16 %) for CHVmP-BV and an ARDI of 92 % (SD 13 %) for CHOP, 88 % (SD 13 %) for ACVBP and 91 % (SD 16 %) for CHVmP-BV, respectively. 10 % of the pts dit not receive an ARD > 80 % (CHOP: 12 %, ACVBP: 2 % and CHVmP-BV: 6 %) and 16.5 % of pts had an ARDI below 80 % (CHOP: 15 %, ACVBP: 26 % and CHVmP-BV: 11 %). Overall, 45 % of the pts had dose modifications (dose delays (DD), dose reductions (DR) or both). Hematological toxicity was the major reason for CT-adjustment. In total, 56 % of pts received granulocyte colony-stimulating factor (G-CSF) treatment (CHOP: 47 %, ACVBP: 100 %, CHVmP-BV: 61 %). The different regimens used having a very different dose-intensity, analysis of effect of ARDI on survival was restricted to the 210 patients treated with CHOP-21. Mean overall survival of these patients according to ARDI category (> 90 %, £ 90 %) was respectively (mean ± SD) 5.38 y ± 0.58 and 2.24 y ± 0.30. Cox regression for effect of ARDI on overall survival was significant (p = 0.002).

Conclusions: (1) With respect to dose-intensity, lymphoma pts treated in hematological departments in Belgium benefit from optimal treatment. (2) CHOP-21 is the most frequently used CT regimen, followed by ACVBP and CHVmP-BV. (3) In pts treated with CHOP-21, overall survival was significantly improved when ARDI was superior to 90 %.
Peripheral Blood Stem Cells vs Bone Marrow for Matched Sibling Transplant in AML and ALL in First Remission.
Authors: Kirschbaum Mark , O’Donnell M. , Spielberger R. , Bhatia R. , Pullarkat V. , Stein A. S. , Kogut N. , Bolotin E. , Chang K. , Land J. , Sarkodee-Adoo C. , Snyder D. , Forman S. J.
Published: October 2019

We report a retrospective comparison of peripheral blood (PBSC) versus bone marrow derived stem cells for allogeneic transplant in a cohort of similarly treated patients based on eight year data from a single institution. Over the period 1995 to 2003, a total of 151 patients with AML or ALL in first CR underwent allogeneic matched sibling donor transplant, 98 AML patients and 53 with ALL. The source of stem cells was bone marrow in 69, G-CSF mobilized peripheral blood stem cells (PBSC) in 82. Of the AML patients, 40 (58%) received marrow, 58 (71%) received PBSC. Of the ALL patients, 29 (42%) received marrow, 24 (29%) got PBSC. The age of patients receiving marrow was 32.7 ± 13.6, and for those getting PBSC was 36.1 ± 16.1. The conditioning regimen was fully ablative (TBI/VP-16) in all but five patients (Fludarabine/Melphalan in 1 marrow and 4 PBSC pts). GVHD prophylaxis was with cyclosporine/methotrexate in patients receiving ablative conditioning, and CSP/MMF in the flu/mel patients.

The time to achieving an ANC >1000 was significantly shorter (P value=.0001) in the PBSC group, 18.2 ±6.6 days vs. 27.2 ±48.7 in the marrow group. Time to achieving a platelet count greater than 25K was significantly shorter (P value=2x10−11) for the PBSC group, 20.9 ± 8.5 vs. 31.8 ± 11.2 for marrow. This was true as well for time to reach platelet counts greater than 100K (P value=10−8), 29.7 ± 28 days for PBSC, 55.8 ±55.6 for marrow. In fact there was a significant increase (P=.0001) in the number of patients, 29 (42%) of marrow recipients vs 11 (13%) of PBSC, who did not at any time achieve a platelet count of >100K. There was no significant difference in survival at 2 years or 4 years between marrow or PBSC-Kaplan-Meier survival probability at 2 and 4 years respectively for AML was.66 and.54 for marrow, and.67 and.54 for PBSC. In ALL, the 2 and 4 year K-M survival probability was.72 and.65 for marrow;.69 and.69 for PBSC recipients. There was no significant difference in acute GVHD, with grade I-II described in 19 (28%) of the marrow pts, 29 (35%) in the PBSC pts, and grade II-IV in 8 (12%) marrow pts and 11 (13%) receiving PBSC (p value =.57), or chronic GVHD (P value=.62). Interestingly, in a subset analysis of patients who were diagnosed with acute GVHD, there was a significant difference in disease free survival at 5 years (see graph), suggesting that there is a qualitative difference between acute GVHD observed as a result of bone marrow versus PBSC as a source of stem cells.

Figure Figure
Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) - EBMT Risk Score Evaluation for Chronic Myeloid Leukemia in Brazil.
Authors: De Souza Carmino , Vigorito Afonso C. , Ruiz Milton A. , Nucci Márcio , Dulley Frederico L. , Fincke Vaneusa M. , Tabak Daniel , Azevedo Alexandre M. , Byington Rita , Macedo Maria C.M. , Saboya Rosaura , Aranha Francisco J.P. , Oliveira Gislaine B. , Zulli Roberto , Miranda Eliana C.M. , Azevedo Wellington M. , Lodi Fernanda M. , Voltarelli José C. , Simões Belinda P. , Colturato Vergílio , De Souza Mair P. , Silla Lúcia , Bittencourt Henrique , Ruiz Lilian P. , Maiolino Angelo , Gratwohl Alois , Pasquini Ricardo
Published: October 2019

This is a retrospective analysis of 1084 patients who received an allogeneic HSCT in 10 Brazilian Centers, from February 1983 to March 2003, aiming to validate the EBMT risk score. Data from transplanted patients and donors regarding patients age, disease stage at transplantation, HLA full-match sibling donor or full-match unrelated donors, donor-recipient gender match and the interval from diagnosis to transplantation, were used as variables to calculate the EBMT risk score. This score was analyzed using Cox Proportional Hazards Model. The OS, DFS, TRM and relapse were analyzed using Kaplan-Meier and cumulative incidence, whenever appropriate. In all there were 647 (60%) males and 437 (40%) females, the median age was 32 years old (range 1 – 59); 898 (83%) were in chronic phase, 146 (13%) were in accelerated phase and 40 (4%) were in blast crisis; 151 (14%) were younger than 20 years old, 620 (57%) were between 20 and 40 and 313 (29%) were older than 40; 1025 (95%) received HLA full-match sibling transplant and only 59 (5%) received an unrelated transplant. Female donor to male recipient occurred in 283 (26%) transplants. The interval from diagnosis to transplantation was less than 12 months in 223 (21%) cases and greater in 861 (79%). The OS, DFS, TRM and, relapse were 49%, 50%, 45%, 25%, respectively. The risk score 0–1 occurred in 179 (17%), score 2 in 397 (37%), score 3 in 345 (32%), score 4 in 135 (12%), and score 5–6 in 28 (2%). The risk scores 0–1 and 2 did not show any difference in terms of OS (58% and 55%, respectively) but they were significantly better than scores 3 or more (score 3 – 44%, 4 – 36 % and, 5-6 - 27%, respectively) (P<0.001). DFS and TRM beyond score 3 were 46%, 49%, respectively and the relapse rate beyond score 5–6 was 77%. Disease status had a negative impact on all outcomes (OS, DFS, TRM, and relapse). OS for female donor - male recipient was 40% compared to 52% for the other patients (P=0.004). DFS and TRM were significant for disease phase and female donor-male recipient (P<0.001 and P<0.003, respectively). In our experience, age and interval from diagnosis and transplant did not show any difference in terms of OS, DFS, TRM, and relapse rate. Our results confirm the usefulness of the EBMT risk score for point-decision in the Imatinib era.
P38 MAPK Inhibition Enhances PS-341 (bortezomib)-Induced Cytotoxicity Against Multiple Myeloma Cells.
Authors: Hideshima Teru , Podar Klaus , Chauhan Dharminder , Ishitsuka Kenji , Mitsiades Constantine , Tai Yu-Tzu , Hamasaki Makoto , Raje Noopur , Hideshima Hiromasa , Schreiner George , Nguyen Aaron , Navas Tony , Munshi Nikhil C. , Richardson Paul G. , Higgins Linda S. , Anderson Kenneth C.
Published: October 2019

PS-341 (pyrazylcarbonyl-Phe-Leu-boronate, bortezomib, Velcade™) represents a class of peptide boronate proteasome inhibitors which inhibit 26S proteasome activity, and demonstrated promise as potential novel anti-cancer therapies. PS-341: induces heat shock proteins (Hsps); activates JNK; induces apoptosis in MM cells mediated by activation of caspase-8, -9, and -3; induces cleavage of DNA protein kinase catalytic subunit (DNA-PKcs) and ATM; overcomes conventional drug resistance conferred by IL-6 or adherence to bone marrow stromal cells (BMSCs); and further abrogates IL-6-triggered signaling cascades by caspase-dependent downregulation of gp130 (CD130). Importantly, a phase II clinical trial of Velcade in refractory relapsed MM demonstrated 35% responses, including 10% complete and near complete responses; however, 65% of patients did not respond to PS-341. We have previously shown that heat shock protein (Hsp)27 is upregulated after PS-341 treatment, that overexpression of Hsp27 confers PS-341 resistance, and that inhibition of Hsp27 overcomes PS-341 resistance. Since Hsp27 is a downstream target of p38 mitogen-activated protein kinase (MAPK)/ MAPK-mitogen-activated protein kinase-2 (MAPKAPK2), we hypothesized that inhibition of p38 MAPK activity could augment PS-341 cytotoxicity by downregulating Hsp27. Although p38 MAPK inhibitor SCIO-469 (Scios Inc, CA) alone did not induce significant growth inhibition in MM.1S cells, it blocked baseline and PS-341-triggered phosphorylation of p38 MAPK, as well as upregulation of Hsp27, associated with enhanced cytotoxicity. Importantly, SCIO-469 enhanced phosphorylation of c-Jun NH2-terminal kinase (JNK) and augmented cleavage of caspase-8 and poly (ADP)-ribose polymerase (PARP). Moreover, SCIO-469 downregulated PS-341-induced increases in G2/M phase cells, associated with downregulation of p21Cip1 expression. These results were further confirmed using transient transfection with p38 MAPK siRNA. Importantly, SCIO-469 treatment augmented cytotoxicity of PS-341 even against PS-341 resistant cell lines and patient MM cells. These studies therefore provide the framework for clinical trials of SCIO-469 to enhance sensitivity and overcome resistance to PS-341, thereby improving patient outcome in MM.
Two Novel Activating Germline Mutations of the C-RAF Proto-Oncogene Predisposing to Solid Tumors and Therapy-Related Acute Myeloid Leukemia.
Authors: Zebisch Armin , Staber Philipp B. , Fischereder Katja , Bodner Claudia , Hiden Karin , Linkesch Werner , Auner Holger W. , Emberger Werner , Windpassinger Christian , Schimek Michael G. , Hoefler Gerald , Troppmair Jakob , Sill Heinz
Published: October 2019

Constitutive activation of the RAS-RAF-MEK-ERK signaling cascade plays an important role in the pathogenesis of AML. We were interested whether abnormalities of the serine-threonine kinases A -, B, - and C -RAF occur in AML and whether they might be causative for activation of this pathway. Expression of these genes was assessed by real-time quantitative (rtq) PCR in 102 patient samples of AML, three AML cell lines and normal CD34+ cells of seven individuals. Results were correlated with the phosphorylation status of ERK 1/2, as measured by immunoblot analysis. 45/82 (54,9%) of AML cases showed ERK phosphorylation indicating constitutive activation of the pathway. However, there was no correlation with expression levels of either A-, B- or C-RAF. Deletions of the B-RAF gene as revealed by rtq PCR were observed in 20/99 (20%) of AML cases and were consistent with cytogenetic results but did not correlate with ERK activity, either. In addition, we screened for mutations of B- and C-RAF in all samples showing ERK phosphorylation using PCR and sequence analysis. Two novel C-RAF missense mutations, both of them located in the kinase domain, were detected in the subgroup of therapy related AML (t-AML): S427G in one and I448V in another patient. Expression of the mutated proteins in mammalian cells resulted in the constitutive activation of MEK and ERK, thus strongly suggesting that the mutations activated C-RAF. Sequence analysis of the primary tumors (colon cancer and teratoma, respectively) as well as buccal epithelial cells revealed these mutations of germline origin and denaturing high pressure liquid chromatography revealed a frequency of <1/400 alleles in the healthy population. Immunohistochemistry using antibodies against phosphorylated ERK demonstrated the pathway to be constitutively activated in the primary tumors as well but inactive in non-neoplastic tissue. In conclusion, we have identified the first activating missense mutations of the C-RAF proto-oncogene in a human tumor. Since both are of germline origin and the primary as well as secondary malignancies showed constitutive activation of the RAS-RAF-MEK-ERK pathway, these mutations might be regarded as predisposing factors in these individuals.
Identification of Cooperating Genetic Alterations in RUNX Leukemia Using the Mouse Model for the Human Familial Leukemia, FPD/AML.
Authors: Osato Motomi , Yamashita Namiko , Yanagida Masatoshi , Huang Liqun , Kogan Scott , Iwasaki Masayuki , Nakamura Takuro , Shigesada Katsuya , Asou Norio , Ito Yoshiaki
Published: October 2019

The RUNX1/AML1 gene is essential for hematopoiesis and is most frequently involved in human leukemias. Loss-of-function of RUNX1 is the common underlying mechanism of RUNX leukemias: those carrying t(8;21), inv(16) and RUNX1 point mutations. As the weakest mode, haploinsufficiency of RUNX1/AML1 causes familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). Mice heterozygous for Runx1 null mutation, Runx1+/−, appear genetically equivalent to human FPD/AML patients, but showed no apparent defects and did not develop spontaneous leukemia, implying that additional genetic alterations are required for leukemia. To identify such abnormalities, retroviral insertional mutagenesis was employed by using BXH2 mice that have spontaneously transmittable ecotropic retrovirus. BXH2-Runx1+/− mice showed a shorter disease latency and more myeloid-specific phenotype than wild-type littermates. Therefore, BXH2-Runx1+/− mice appear to serve as the mouse model for FPD/AML to investigate myeloid leukemia. The genes selectively affected in BXH2-Runx1+/− mice by retroviral integration are likely to cooperate with Runx1+/−. c-Kit locus was involved twice in 24 BXH2-Runx1+/− but neither in 17 wild-type littermates nor in 135 inbred BXH2 mice reported in the RTCGD database, suggesting that c-KIT mutation cooperates with RUNX alteration in human leukemia. In fact, constitutively active mutants of c-KIT or functionally equivalent FLT3, were found in 10 out of 25 (40%) of human RUNX leukemias. Together, BXH2-Runx1+/− system appears useful to identify the genes cooperating with RUNX1 abnormalities.
Sensitization for γ-Irradiation-Induced Apoptosis by Smac.
Authors: Giagkousiklidis Stavros , Kasperczyk Hubert , Vogler Meike , Debatin Klaus-Michael , Fulda Simone
Published: October 2019

Smac is released from mitochondria during the onset of apoptosis and promotes apoptosis via abrogating the binding of Inhibitor of Apoptosis Proteins (IAPs) to caspases. γ-irradiation is one of the most commonly used therapeutic approaches in clinical oncology, which triggers cell death in tumors via DNA and/or membrane damage. Since we recently found that Smac agonists sensitized even resistant tumors for apoptosis induced by death receptor ligation or anticancer drugs, we investigated the effect of Smac agonists on apoptosis following γ-irradiation in the present study. Here, we report for the first time that overexpression of mitochondrial or cytosolic Smac significantly increased radiosensitivity of various cancers.

Transfection-enforced expression of Smac strongly enhanced apoptosis upon γ-irradiation in SH-EP neuroblastoma cells, which were resistant to g-irradiation in the absence of Smac. Importantly, Smac overexpression also reduced clonogenic tumor cell survival following γ-irradiation. Analysis of signaling pathways revealed that overexpression of Smac resulted in more rapid and more potent activation of caspase pathways, e.g caspase-2, -3,- 8, -9. The broad range caspase inhibitor zVAD.fmk abrogated apoptosis upon γ-irradiation indicating that apoptosis was mediated by caspases. In addition, overexpression of Smac promoted loss of mitochondrial membrane potential and cytochrome c release upon γ-irradiation. Interestingly, γ-irradiation-induced mitochondrial perturbations were blocked in the presence of the caspase inhibitor zVAD.fmk suggesting that caspase activity was required for mitochondrial alterations in response to γ-irradiation. Notably, cell cycle alterations and activation of NF-κB occured in a similar manner in vector control and Smac-transfected cells suggesting that Smac did not significantly alter the initial cellular stress response upon γ-irradiation. Importantly, Smac overexpression sensitized various tumor cell lines for γ-irradiation-induced apoptosis, indicating that the sensitization effect of Smac for γ-irradiation was not restricted to a particular cell type. By demonstrating that Smac can sensitize various tumor cells towards γ-irradiation-induced cell death, our findings provide for the first time evidence that Smac agonists may be a useful tool to enhance radiosensitivity in a variety of human cancers.
Engraftment of NOD/SCIDγcnull Mice with Multilineage Neoplastic Cells from Patients with Juvenile Myelomonocytic Leukemia.
Authors: Yamamoto Tomoko , Nakamura Yoichi , Ito Masafumi , Xu Yinyan , Yagasaki Hiroshi , Kamachi Yoshiro , Kudo Kazuko , Kojima Seiji
Published: October 2019

Jevenile myelomonocytic leukemia (JMML) is a rare myeloproferative disorder of early childhood, arising from pluripotent stem cells. Previous studies indicate the clonal nature of JMML, involving myeloid, erythroid, megakaryocyte and B-lymphoid lineage. However, it is unclear whether T lymphocyte lineage is involved or not. In one previous report, a patient with JMML and cytogenetic evidence of monosomy 7, a combination of fluorescence activated cell sorting and fluorescence in situ hybridization (FISH) analysis confirmed no T cell lineage involvement. One explanation of sparing T cell lymphocyte involvement in JMML is that the majority of T-lymphocytes are long-lived and borne before the occurrence of the disease. We demonstrated that cells from six patients of JMML repopurate in NOD/SCIDγcnull mice and differenciate into granulocyte, monocyte, erythroid, B-lymphocyte, T-lymphocyte and natural killer(NK) cells. The diagnosis of JMML was based on the internationally accepted criteria. 0.01 to 1×107mononuclear cells were injected through the tail vein of five week old NOD/SCIDγcnull mice. 12 week after transplantation mice were sacrificed and the cells are collected from the femoral bone and spleen and subjected to flow cytometry. The percentage of human CD45 antigen positive cells ranged from 41 to 73%. To examine the involvement of lymphocyte subpopulations, we purified human CD3+,CD19+and CD56+ cells from a murine bone marrow and spleen cells transplanted from a patient with monosomy 7. The purity of cells were more than 95%. The percentage of monosomy 7 cells determined by FISH analysis ranged from 96 to 100% in purified CD3+, CD19+and CD56+ cells. These findings support the concept that JMML originate in transplantable multilineage hematopoietic stem cells. This novel murine xenotransplant model should be useful to investigate the nature of stem cells and test new therapies for patients with JMML.
3D, a Novel Mutation That Confers Defective Sensing by Toll-Like Receptors 3, 7 and 9.
Authors: Tabeta Koichi , Du Xin , Philippe Georgel , Hoebe Kasper , Mann Navjiwan , Mudd Suzanne , Huser Nanni , Shamel Louis , Crozat Karine , Tarantino Lisa , Cooke Michael , Beutler Bruce
Published: October 2019

The mammalian Toll-like receptors (TLRs) comprise a key interface between cells of the host and all classes of microbial pathogen. By sensing nucleic acids, the endosomal TLRs 3, 7, and 9 play a particularly important role in the detection of viral infection, and permit the host to mount an immediate and efficacious anti-viral response. In an effort to identify novel components of the TLR signaling apparatus, we have pursued a program of germline saturation mutagenesis with N-ethyl-N-nitrosourea (ENU) in mice. The germline mutants are produced on the C57BL/6 background, and peritoneal macrophages from individual animals are screened for their competence to respond to TLR-dependent microbial inducers. A strong phenodeviant called 3d was identified in the F3 generation as a non-responder to nucleoside-based molecules such as unmethylated CpG oligodeoxynucleotides, Resiquimod (a drug of the imidazoquinoline class) and Poly I:C. TNFa production induced by TLRs 3, 7, and 9 was completely prevented in 3d homozygotes. However, heterozygotes were unaffected, and the ability to produce TNFa in response to other bacterial compounds, sensed by TLRs 1, 2, 4, and 6, was intact. 3d homozygotes show extreme susceptibility to infection by mouse cytomegalovirus (MCMV) infection in vivo. After IP inoculation with MCMV (104 PFU), 3d mutants showed impaired production of type I and type II interferons, TNF, and IL-12 in serum (p<0.001). With larger inocula (5 x 105 PFU) a splenic viral titer approximately ten-thousand fold higher than that in controls (p<0.05) and rapid mortality (p<0.0001) were observed. This novel recessive phenotype is fully penetrant on the C57BL/6 and C3H/HeN x C57BL/6 hybrid backgrounds. The 3d mutation was mapped to mouse chromosome 19 using a panel of microsatellite markers and at present, has been confined to 0.4 a Mbp critical region on 3000 meioses. 3d appears to encode a protein essential for TLR-mediated detection of nucleic acids, which is indispensable for effective innate defense against viral infection.
Killed but Metabolically Active Recombinant Listeria monocytogenes as an Antigen Delivery and Activation Platform for Human Dendritic Cell-Based Cancer Immunotherapy.
Authors: Brockstedt Dirk G. , Skoberne Mojca , Yewdall Alice , Bahjat Keith S. , Corash Larry , Dubensky Thomas W. , Bhardwaj Nina
Published: October 2019

Dendritic cells (DCs) are bone marrow-derived professional antigen presenting cells which play a pivotal role in initiating immune responses. To achieve optimal potency for priming of naive T cells, DCs must not only present high levels of MHC peptide complexes, but must also undergo maturation. An ideal DC-targeting vaccine formulation, therefore, should simultaneously deliver multiple antigens and activate DCs to increase expression of surface MHC-peptide complexes, costimulatory molecules, and production of immuno-stimulatory cytokines. Despite the development of a variety of ex vivo methods, no standard efficient and cost effective methods combining antigen loading and activation/maturation of DCs for clinical use currently exist. Here we report the development of a novel DC antigen-loading platform based on killed, but metabolically active (KBMA) recombinant Listeria monocytogenes (Lm). While recombinant Lm has a relatively long history as a cancer vaccine platform, its utility as a DC activation and loading platform has not been fully explored. KBMA Lm were 8-logs more sensitive to photochemical inactivation by the combined treatment of S-59 psoralen (amotosalen HCl) and long-wave ultraviolet light (S-59/UVA), by virtue of abrogation of nucleotide excision repair (NER) capacity, through engineered deletion of the bacterial UvrAB genes. Due to random distribution of infrequent psoralen adducts, KBMA Lm cannot propagate, but are metabolically active and can express their genetic repertoire, and program presentation of expressed heterologous antigens via the MHC class I and class II pathways. Infection of human monocyte-derived or blood myeloid DCs with KBMA Listeria induced their maturation and production of pro-inflammatory and pro- Th1 cytokines. The DCs achieved a fully mature phenotype, comparable to that induced by the standard maturation stimulus MCM-mimic. To determine whether KBMA Lm could program presentation of encoded antigens by the MHC class I pathway, we constructed a recombinant Lm vaccine expressing CD8+ T cell epitopes of influenza matrix protein (MP58–66) and MelanA/Mart-1 protein (Mart126–35). DCs infected with the recombinant KBMA Lm vaccine presented the recombinant class I epitopes and induced IFN-γ production by MP58–66- or Mart126–35-specific human CD8+ T cell clones. These studies demonstrate that KBMA Listeria is a novel and powerful ex vivo DC antigen loading platform combining a potent DC-maturation stimulus with the cytosolic delivery of recombinant antigen for presentation.
Anti-Tumor Activity of a Novel Immunosuppressant FTY720 in Multiple Myeloma.
Authors: Yasui Hiroshi , Hideshima Teru , Roccaro Aldo M. , Shiraishi Norihiko , Hamasaki Makoto , Ishitsuka Kenji , Kumar Shaji , Raje Noopur , Li Guilan , Tai Yu-Tzu , Podar Klaus , Chauhan Dharminder , Anderson Kenneth C.
Published: October 2019

Sphingosine and its metabolites are bioactive sphingolipids involved in lipid biosynthesis, signal transduction and apoptosis. FTY720, a synthetic sphingosine analogue of myriocine derived from culture filtrates of Isaria sinclairii, has been reported to interact with the sphingosine-1-phosphate specific G protein-linked receptors (S1P1, 3, 4 and 5) (Mandala S et al. Science, 2002) and alter the migration and homing of lymphocytes, thereby inhibiting the immune response (Matloubian M et al. Nature, 2004). Recent studies have also shown that FTY720 induces growth inhibition and/or apoptosis in human cancer cells in vitro as well as in vivo murine model (Azuma H et al. Cancer Research, 2002). To date, however, the biologic sequelae of inhibiting sphingosine-1-phosphate activity on multiple myeloma (MM) cells have not been demonstrated. In the present study, we examined whether FTY720 triggers anti MM activity. FTY720 induced potent cytotoxicity against MM cell lines including MM.1S, U266, RPMI8226, with IC50 at 24 h of 3.0 – 7.0 mM, assessed by trypan-blue exclusion and MTT assays. FTY720 also inhibited growth of doxorubicin (Dox)-resistant RPMI8226-Dox40 and dexamethasone (Dex)-resistant MM.1R cell lines, with IC50 values similar to the parental drug-sensitive cell lines. In contrast, no cytotoxicity of FTY720 was recognized against human peripheral blood mononuclear cells from normal healthy donors. The combination of Dex with FTY720 demonstrated enhanced cytotoxicity compared to either agent alone. Importantly, neither interleukin-6 (IL-6) nor insulin like growth factor-I (IGF-I), which induces MM cell growth and protection against Dex-induced apoptosis, protected against FTY720-induced growth inhibition. The anti-MM mechanisms of action of FTY720 were next studied, and FTY720 induced caspase-dependent apoptosis in MM cell lines: FTY720 triggers caspase−8, −9 and −3 cleavage, followed by PARP cleavage and DNA fragmentation, as confirmed by Western blotting and agarose gel electrophoresis, respectively. Moreover, FTY720 abrogated both IL-6 mediated phosphorylation of Akt-1, STAT3 and p42/44MAPK, and IGF-I mediated Akt-1 phosphorylation. Importantly, paracrine MM cell growth with bone marrow stromal cells was strongly inhibited by FTY720. These results suggest that FTY720 overcomes drug resistance in MM cells and, providing the rationale for its clinical evaluation to improve patient outcome in MM.
Comparison of Na18F and 18F-Deoxyglucose Positron Emission Tomography in Patients with Multiple Myeloma.
Authors: Troncy Jacques , Mercier Sylvie , Roy Pascal , Michallet Mauricette , Dumontet Charles M. , Janier Marc
Published: October 2019

Staging of patients with myeloma currently takes into account the number of bone lesions identified with standard X-rays. Magnetic Resonance Imaging (MRI) appears to be a more sensitive imaging technique in patients with multiple myeloma (MM) but it is not adapted to the investigation of the entire skeleton. Positron Emission Tomography (PET) imaging appears promising but this approach remains to be validated in patients with MM. Most patients with neoplasia have been performed with 18F-2-deoxy-D-glucose (FDG), a functional marker suggested to identify “active” tumor sites and useful to assess bone or extra-osseous tissue. Conversely Na18F binding is strictly limited to bone. To determine the potential usefulness of these two markers we performed a comparative analysis of these two imaging techniques in patients with MM.

Nine patients with progressive MM, including bone lesions, underwent x-ray imaging, MRI, 18FDG and Na18F scanning. Both PET scans were performed within a one-week time frame in each patient. The number and localizations of suspect lesions identified by each type of imaging technique was assessed independently then compared. 18FDG and Na18F data were compared to X-ray/MRI data for skull, spine, pelvis and femur/humerus localizations. Localizations were considered positive in case of increased fixation with 18FDG or increased or decreased fixation with Na18F.

Comparison of FDG data with X-ray/MRI showed that FDG allowed the detection of only 3 of 35 types of localizations identified by X-ray/MRI, whatever the localization (skull, femur/humerus, spine and pelvis) observed on X-ray/MRI. 18FDG showed three lesions which had not been identified by X-ray/MRI, as well as 8 costal localizations and 1 extra-osseous localization. Comparison of Na18F data with X-ray/MRI showed that Na18F allowed the detection of 17 of 35 types of localizations. In particular, Na18F identified 13 of the 19 spinal localizations observed with x-ray/MRI but only 2 of 9 skull lesions. Na18F identified 11 localizations which had not been identified by X-ray/MRI. Furthermore Na18F also identified costal localizations in all patients. Qualitative analysis of PET scans showed that background was significantly lower with Na18F imaging than with 18FDG imaging. In conclusion 18FDG and Na18F provide significantly different results in patients with progressive MM. 18FDG imaging was poorly correlated with X-rayMRI while Na18F imaging was particularly useful to identify costal and spinal lesions. PET scan could be a useful method in the staging of MM patients at diagnosis or to determine response.

Figure Figure
The Effect of a Toll Receptor-7 Agonist (S28690) on the Immunogenicity of Chronic Lymphocytic Leukemia Cells.
Authors: Hicks Lisa , Shi Y. , Mena J. , Hammond C. , Tomic Y. , Tomai M. A. , Miller R. L. , Spaner D. E.
Published: October 2019

Background: Chronic lymphocytic leukemia (CLL) cells are weakly immunogenic, a property that may contribute to disease progression and inhibit the effectiveness of immunotherapies such as vaccines. Low surface expression of co-stimulatory molecules contributes to this poor immunogenicity. CLL cells express Toll-Like-Receptor-7 (TLR7), a powerful modulator of innate immunity. TLR7 agonists may be capable of enhancing the immunogenicity of CLL cells and thereby increasing T cell mediated killing of CLL.

Methods: Circulating CLL cells were isolated directly from consenting patients by negative selection. TLR7 mRNA expression by CLL cells was demonstrated by RT-PCR. CLL cells were then incubated with S28690 (a TLR7 agonist), or with a negative control for 24–72h. Expression of the costimulatory molecules CD80, 83, 86, and 54 was determined by flow cytometry pre and post-incubation. Experiments were repeated in the presence of a NFkB inhibitor (dexamethasone), a p38 MAPK inhibitor, and a protein kinase C agonist (PDB). The effects of S28690 on phosphorylated-IkB and phosphorylated-STAT3 levels were measured by immunoblotting. The capacity of S28690-incubated CLL cells to stimulate T cell proliferation and killing was determined in mixed lymphocyte responses.

Results: All tested CLL samples (n=20) expressed TLR7 mRNA, while Jurkat cells (T cell origin) did not. After incubation with S28690, CD80, 83, 86, and 54 surface expression increased on all CLL samples tested. The relative increase varied from 4 to 9-fold and was positively correlated with CD38 expression. NF-kB and p38 inhibitors decreased the effects of S28690 on co-stimulatory molecule expression while PDB amplified the effect. After incubation with S28690, IkB and STAT3 phosphorylation increased in CLL cells. S28690-incubated-CLL cells were able to stimulate moderate T cell proliferation, but did not increase T cell mediated killing of CLL cells. However, CLL cells incubated with both S28690 and PDB (a PKC agonist), exhibited much lower amounts of phosphorylated STAT3, triggered marked T cell proliferation, and stimulated T cell mediated killing of CLL cells.

Conclusions: S28690 (a TLR7 agonist) causes increased expression of co-stimulatory molecules by CLL cells in vitro and transforms CLL cells into moderate stimulators of T cell proliferation. The effects of S28690 are synergistic with PKC agonists, potentially as a result of S28690-mediated NFkB activation and concurrent PKC-mediated inhibition of STAT3. These findings may find clinical application in immunotherapeutic approaches to CLL.
The Sesquiterpene Lactone Parthenolide Rapidly and Selectively Induces Apoptosis of B-Chronic Lymphocytic Leukemia Cells.
Authors: Steele Andrew J. , Casanova Anouska A. , Hoffbrand A. Victor , Mehta Atul B. , Ganeshaguru Kanagasabai , Jones Dylan T. , Wickremasinghe R. Gitendra
Published: October 2019

B-chronic lymphocytic leukaemia (CLL) is the most frequent leukaemia in the western world. CLL is incurable using conventional therapy. While the disease is controlled by treatment with chlorambucil (CHL) or fludarabine, extended exposure to these agents results in drug resistance. It is therefore important to identify novel agents which rapidly and selectively induce apoptosis of cells isolated from patients who have become resistant to conventional drugs. The sesquiterpene lactone parthenolide (PTL) is the active component of several medicinal plants which have been used for hundreds of years for the treatment of migraines, pain and inflammatory conditions. In some cell lines, PTL has been shown to block components of the NFκB pathway including the IκB kinase complex and/or the p65 subunit of NFκB. Here we investigated the sensitivity of isolates from 25 CLL patients (representing stages A, B and C) to PTL (1.25–80 μM) and CHL (3.125–200μM). IC50 values were evaluated by the 4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay. Apoptosis induction was quantified by FACScan analysis of annexin V binding and by western blot detection of cleavage of the caspase 3 substrate poly (ADP ribose) polymerase (PARP). CHL resistance varied across the cohort from highly sensitive to highly resistant, with IC50 values in the range 5.3 to >200 μM. In contrast, all isolates tested were sensitive to PTL, with IC50 values in the range 3 to 20 μM. Strikingly, isolates from extensively-treated patients which were highly resistant to elevated levels of CHL in vitro were sensitive to PTL (Figure 1). PTL induced very rapid PARP cleavage which became clearly evident between 3–6 h of exposure to 5 μM PTL. In contrast, PARP cleavage was not detected until >12h exposure to 75 μM CHL in isolates which were sensitive to this drug. Furthermore, pulse/wash-out experiments showed that a very brief (1-2h) exposure to PTL was sufficient to induce commitment to the apoptotic programme. The annexin V-binding assay revealed that CLL cells were strikingly more sensitive to apoptosis induction by 2–5 μM PTL than were normal T or B lymphocytes or CD34+ bone marrow progenitor cells (Figure 2). Western blot analysis revealed that PTL-induced PARP cleavage was preceded by downregulation of anti-apoptotic mcl-1, a key mediator of the survival of B lineage lymphoid cells. However, we failed to detect a PTL-induced decrease in IκBαphosphorylation, suggesting that inhibition of IκB kinase did not contribute to the mechanism of CLL cell killing. In conclusion, the rapid, selective action of PTL and its ability to kill CLL isolates refractory to cytotoxic drugs suggest that this agent may be of value in the treatment of CLL patients who have become resistant to conventional therapeutic regimes.

Figure Figure
Sequential Phase II Studies of Flavopiridol by 72-Hour Continuous Infusion and 1-Hour Intravenous Bolus for the Treatment of Relapsed B-Cell Chronic Lymphocytic Leukemia: Results from CALGB Study 19805.
Authors: Byrd John C. , Peterson Bercedis L. , Gabrilove Janice L. , Odenike Olatoyosi M. , Grever Michael R. , Rai Kanti R. , Vardiman James W. , Larson Richard A. ,   Cancer and Leukemia Group B
Published: October 2019

Flavopiridol has in vitro activity in CLL and promotes apoptosis independent of p53 function or prior fludarabine exposure. We sought to determine if flavopiridol administered using two different schedules has activity in CLL. Patients with previously treated CLL were enrolled on two sequentially performed phase II studies. Patients in the first trial received flavopiridol (50 mg/m2 daily) as a continuous infusion (CI) over 72-hours every 2 weeks. Patients in the second trial received flavopiridol 50 mg/m2 as a 1-hour intravenous bolus (IVB) daily for three days repeated every 3 weeks. Patients received up to 6 (CI cohort) or 8 (IVB cohort) cycles of therapy. Fifteen patients enrolled in the 72-hour CI phase II trial; 6 (40%) had intermediate (Rai stage I or II) and 9 (60%) high (Rai stage III and IV) risk stages. No responses were noted in this group with 27% having stable disease (SD) and 73% progressive disease (PD). Thirty-six patients enrolled in the IVB study, with 13 (36%) having intermediate and 23 (64%) having high-risk disease. Four patients (11%) had partial responses, 19 (53%) SD, and 13 (36%) PD. The progression-free survivals for responders in the IVB study were 2.9, 3.2, 8.7, and 19.3 months. The median progression-free survival was 2.1 months (95% confidence interval [CI] 1.8 – 3.8) for patients in the CI study and 3.2 months (95% CI [2.5 – 7.4]) for the IVB study. The median overall survival was 27 months (95% CI [20–42]) for the CI study and 24 months (95% CI [18–31]) for the IVB study. Toxicity was manageable and included mainly myelosuppression (granulocytopenia and thrombocytopenia), infections, diarrhea and fatigue. Grade 3 and 4 toxicities were 20% and 27%, respectively, on the CI study and 39% and 33% on the IVB study. One patient on the IVB study had tumor lysis syndrome that was managed medically and did not require dialysis. There was one on-study death following a myocardial infarction on the IVB study. We conclude that flavopiridol has modest, schedule-dependent clinical activity in relapsed CLL and warrants future investigation utilizing alternative schedules of administration.
Heparin-Induced Thrombocytopenia Antibodies Do Not Predict Adverse Outcomes in End-Stage Renal Disease Patients on Dialysis.
Authors: Asmis Lars M. , Segal Jodi B. , Plantinga Laura C. , Fink Nancy E. , Kerman Jonathan S. , Kickler Thomas S. , Coresh Josef , Gardner Lawrence B.
Published: October 2019

Introduction: Heparin-induced thrombocytopenia (HIT) is a syndrome in which anti-platelet factor 4/heparin antibodies (HITab) and thrombocytopenia develop after heparin exposure. While HIT is an established risk factor for arterial and venous thromboses, HITab, in the absence of thrombocytopenia, may also be a prothrombotic risk factor. As patients with chronic kidney disease on hemodialysis are chronically exposed to heparin, we hypothesized that the presence of HITab may contribute to the high rates of thrombotic disease and mortality in these patients.

Patients and methods: We analyzed data from the CHOICE study, a prospective cohort study of incident dialysis patients. Clinical data were available from medical record review. Arterial cardiovascular events and events of vascular access occlusion were validated by chart review and adjudication. Venous thromboembolic events were defined by ICD-9 codes. Thrombocytopenia was defined as a platelet count <150,000/mm3 or a >50% decline from a previous platelet count. Median duration of follow-up is 2.7 years. We measured HITab using a commercial ELISA assay in stored serum obtained 6 months after cohort enrollment of 740 participants (596 on hemodialysis, 144 on peritoneal dialysis). ELISA results were corrected for inter-plate variability. A positive HITab test associated with thrombocytopenia in the subsequent three months was considered compatible with HIT. We used techniques of logistic regression and of survival analysis with adjustment for potential confounders in our analyses.

Results: 10.3% of all patients were positive for HITab at six months. HITab positivity was not associated with thrombocytopenia. HITab did not predict subsequent outcomes including cardiovascular (hazard ratio (HR) 0.96, 95% confidence interval (CI) 0.65–1.41), vascular access occlusive (HR 0.82, CI 0.40–1.71), or venous thromboembolic events (HR 1.39, CI 0.17–11.5) and did not predict mortality (HR 1.00, CI 0.66–1.53). 9 patients (1.2%) had results compatible with HIT (thrombocytopenia + HITab). 1 patient with HIT (0.13%) had venous thrombosis yielding a hazard ratio of 13.7 (CI 1.63–115.5) for HIT and thrombosis.

Conclusions: HITab seropositivity after six months of dialysis is frequent, but is not associated with thrombocytopenia. In end-stage renal disease patients treated by dialysis HITab seropositivity in the absence of thrombocytopenia is not an independent risk factor for cardiovascular morbidity or mortality. However, HIT may occur in this patient population and is associated with thrombosis.