bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneity of New Hodgkin’s Disease Patients in Terms of Their Quality of Life.
Authors: Novik Andri A. , Ionova Tatiana I. , Kishtovich Anton V. , Uspenskaya Olga S. , Osipova Nina E. , Fedorenko Denis A.
Published: October 2019

Background: It is quite obvious that population of new Hodgkin’s disease (HD) patients is heterogeneous in terms of clinical characteristics. Therefore stratification of patients is necessary to select appropriate treatment regimen. It was shown that new lymphoma patient population is heterogeneous in terms of base-line QoL (Novik A et al, 2003).

The aim of the study was to identify if new HD patients are heterogeneous in terms of QoL and to stratify patients using QoL parameters.

Patients and methods: 114 new HD patients (stage IIB-IV, mean age 30.2 (SD 13.5), males/females - 55/59) were enrolled in the study. Patients filled in EORTC QLQ-C30 at baseline. The method of integral profiles (MIP) was used to calculate integral QoL index (IQL), the coefficient of QoL deformation (CQLD) and the index of symptom severity (ISS). The above indexes were used for stratification by the cluster analysis (Ward method, percent of cases correctly classified - 98.25%). Discriminant analysis was performed for stratification testing.

Results: Three groups of HD patients were identified after stratification. IQL, CQLD and ISS were as follows: subgroup 1 (n=5, mean age 40.8 (22.9), male/female 1/4) − 0.04, 1.7, 0.17; subgroup 2 (n=47, mean age 34.3 (14.5), male/female 23/24) − 0.35, 0.62, 0.09; subgroup 3 (n=62; mean age 26.0 (9.7); male/female 31/31) − 0.74, 0.24, 0.01. Group 1 has the worst QoL and is characterized by low IQL, pronounced symptoms and high deformation of QoL profile. Group 3 has the best QoL with high IQL, weak symptoms and low deformation of QoL profile. Group 2 has intermediate values of above parameters. Differences between groups were significant (p<0.05) by ANOVA.

Conclusion: The method of integral profiles to obtain the integral QoL index and the coefficient of QoL deformation in HD patients is proposed. Stratification of new HD patients by cluster analysis resulted in three patient subgroups according to their QoL data. Identification of subgroups of HD patients on the basis of their QoL has a good potential for better stratification of new HD patients and in doing so might be used for improvement of the intervention strategies.
Overexpression of Mitochondrial Ferritin Causes Cytosolic Iron Starvation and Changes Cellular Iron Homeostatis.
Authors: Nie Guangjun , Sheftel Alex D. , Kim Sangwon F. , Ponka Prem
Published: October 2019

Cytosolic ferritin is well known to sequester and store iron and, consequently, protect cells against iron-mediated free radical damage. However, the function of the newly discovered mitochondrial ferritin (MtFt) is unknown. To examine the role of MtFt in cellular iron metabolism, we established a cell line that stably overexpresses the mouse MtFt under the control of tetracycline. The overexpression of MtFt caused a dose-dependent iron deficiency in the cytosol that was revealed by increased RNA-biding activity of iron regulatory proteins (IRPs) with a concomitant increase in transferrin receptor levels and a decrease in cytosolic ferritin. Consequently, the induction of MtFt resulted in a dramatic increase (2-fold) in cellular iron uptake from transferrin, most of which ended up in MtFt within mitochondria. Similarly, the induction of MtFt caused a shift of iron from cytosolic ferritin to MtFt. Moreover, the cell permeable iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) mobilized approximately twice as much iron from wild-type cells as compared to those overexpressing MtFt suggesting that iron in this protein is less accessible to SIH. Furthermore, the expression of MtFt was associated with a decrease in the activity of both mitochondrial and cytosolic aconitase activity, the latter being in agreement with the increase of IRP binding activity. In conclusion, our results indicate that the overexpression of MtFt causes a dramatic change in intracellular iron homeostasis and that the shunt of iron to mitochondrial storage protein likely limits its availability for functional iron proteins.
DHPLC Scan of Iron Genes in Consecutive Patients with Suspected Iron Overload.
Authors: Girelli Domenico , Biasiotto Giorgio , Foglieni Barbara , Bozzini Claudia , Polotti Anna , Ferrari Francesca , Tenuti Ilaria , Gerardi Gian Mario , Cremonesi Laura , Corrocher Roberto , Arosio Paolo
Published: October 2019

Background: Hereditary Hemochromatosis, once considered a monogenic disorder, is now seen as a polygenic disease, with clinical phenotype also influenced by several environmental factors. Besides the classic HFE gene, other genes involved in modulation of iron homeostasis and clinical phenotype include those coding for hemojuvelin and hepcidin (both responsible for Juvenile Hemochromatosis), ferroportin, and, possibly, H-ferritin.

Methods: we used DHPLC to scan mutations in the above mentioned genes in 55 consecutive patients recently referred to our tertiary care unit for iron overload disorders. Many of them had at least biochemical signs of iron overload not explained, or not completely explained, by classic or rare HFE mutations.

Main results: the −72 C→T variation in the promoter of hepcidin gene, near the putative TATA box, was found in a H63D heterozygous male with unexplained biochemical signs (serum ferritin 660 μg/L, TS 45%), who, rather, should have been protected by several previous blood donations. We recently found this new hepcidin mutation in a family from another cohort (Biasiotto et al., in press): genotype/phenotype correlation data were also consisting with a functional role of this mutation. Two other hepcidin variations were found in this series, the 108 G→A (new), and the 212 G→A. While the first appeared silent, familial study suggests that the second may be functional in association with C282Y. No new or functional variations were found in hemojuvelin or H-ferritin genes. Several polymorphisms were detected in the ferroportin gene, including two new sequence variations (−116 T→C in the promoter; 691+20C C→T in intron 4), that occurred in two unrelated subjects with wild-type HFE genotype. Their functional role is currently under investigation by extensive family studies and/or quantitative evaluation of hepatic iron by MRI/liver biopsy.

Conclusions: DHPLC scan of iron genes appears as a helpful tool for integrating clinical data in selected patients referring for suspected iron overload, as well as for rapid identification of new mutations.
DMT1: Tissue Specific Expression and Alternative Splicing.
Authors: Mims Martha P. , Guan Yongli , Prchal Josef T.
Published: October 2019

Divalent metal ion transporter 1 (DMT1) functions in iron absorption at the apical surface of the duodenal enterocyte and in iron transport out of the endosomal compartment of the erythroblast, however, DMT1 is also expressed in cells which have no apparent role in iron metabolism. At least 4 isoforms of DMT1 mRNA have been identified that contain alternative 5′ (1A, 1B) and 3′ (IRE+, IRE-) exons. Recently we reported the first human mutation of the DMT1 gene in a patient with severe hypochromic/microcytic anemia. This patient was homozygous for a mutation in the ultimate nucleotide of exon 12 which resulted in preferential skipping of exon 12 during processing of pre-mRNA. Amplification of DMT1 mRNA from peripheral blood cells of this patient revealed multiple products; hematopoietic cells from normal controls had a similar, but not identical, pattern. No such additional PCR products were seen when DMT1 mRNA from other tissues or cultured cells was evaluated. To further investigate this observation, we amplified full length (1700–1800 bp) DMT1 transcripts (1A/IRE+, 1A/IRE-, 1B/IRE+ and 1B/IRE-) from a variety of tissues types, cell lines, and from hematopoietic cells. Expression of the 4 isoforms differed between the different cell types. EBV lymphocytes expressed primarily 1B/IRE+ and 1B/IRE- isoforms. Notably, EBV lymphocytes derived from the mutant patient expressed DMT1 mRNA which was 120 bp smaller than DMT1 PCR products from controls, corresponding to skipping of exon 12. Platelets, granulocytes and reticulocytes expressed primarily the 1B/IRE+ isoform, however, unlike the other cell types examined, more than one mRNA species was present (see figure). Full length high molecular weight PCR products derived from these cells were isolated and subcloned. Plasmid DNA was isolated, subjected to digestion with EcoR1, and the DMT1 inserts were sequenced. All inserts began with exon 1B and ended with exon 16 (IRE+); however, none contained “normal” full length DMT1 sequence. The majority contained either truncated portions of exon 3 or inserted portions of intron 3. Utilization of cryptic splice sites within exon 3 or within intron 3 resulted in early stop codons. We propose that alternative splicing of DMT1 mRNA either via skipping of entire exons or via the use of cryptic splice sites may provide a manner for regulating DMT1 activity in hematopoietic cells. Further studies are underway to elucidate this possibility and to evaluate the signals for alternative splicing.

Figure Figure
Age Related Changes in Hoechst 33342 Efflux Dynamics and Side Population Phenotype in Murine Bone Marrow.
Authors: Greenwood Matthew J. , Lansdorp Peter M.
Published: October 2019

The mechanisms underlying the aging of the hematopoietic stem cell (HSC) compartment remain poorly understood. The ATP-binding cassette cell surface transport protein, ABCG2, has been identified as the transporter responsible for Hoechst 33342 (Hst) efflux in primitive stem cells and its expression is associated with the side population (SP) phenotype in both murine and human bone marrow (BM). ABCG2 expression and Hst efflux activity is highest in those cells with the greatest repopulating potential and is progressively downregulated during differentiation. The substrate profile of ABCG2, which includes a number of antineoplastic drugs, protoporphyrin IX and the chlorophyll breakdown product pheophorbide, suggest that ABCG2 transporters may function to protect stem cells from cytotoxic insults, a function which may be of great importance in stem cell maintenance. Amongst laboratory mice, the C57BL/6 strain is the longest lived and appears to accumulate HSC’s with age as assessed by both phenotype and colony forming assays. While the phenotypic features of the SP profile have been well characterized in both humans and young mice, little is known of the Hst efflux dynamics or phenotype of the SP profile in old and very old C57BL/6 mice. In order to further characterize the SP profile in old mice, whole BM was extracted from the femurs, tibiae, pelvis and thoracolumbar vertebral bodies of young (9–13 week) and old (95–108 week) C57BL/6 (Ly5.1) mice. Cells were stained with 5μg/ml Hst followed by staining with a combination of CD45.1 FITC, Sca1 PE, c-kit APC, CD34 FITC, biotinylated CD34 and lineage markers and strep PE-Texas Red. In addition, serial sampling of Hst incubated cells was performed to assess Hst efflux activity at 20 mins incubation through to 100mins. Six-color flow analysis was performed on a FACS Vantage™ (BD) cytometer and data analyzed using FlowJo™ software. Results show a marked increase in cells with an SP phenotype in old vs young mice (mean±SD 1.85%±0.88 vs 0.15%±0.09) which were more highly enriched for CD34-Sca1+ckit+ (22.2%±8.65 vs 8.89%±6.7) cells. Subdividing the SP profile into four regions (R1 to R4) from highest to lowest Hst efflux activity revealed that in old mice, SP cells with the highest Hst efflux activity were almost exclusively of a CD34-Sca1+ckit+ phenotype (82.3%±14.0 vs 11.5%±7.8), with a decreasing proportion of these cells represented throughout the remaining SP tail, though a significant proportion of cells within R4 remain CD34-Sca1+ckit+ (15.3%±7.83 vs 4.19±3.01). Similar patterns have been observed in both whole and lineage depleted BM. In addition, BM cells from old C57BL/6 mice show prolonged Hst efflux activity with an increase in cells in the SP gate at 100 mins (1.51%±0.50 vs 0.10%±0.06). We conclude that in old C57Bl/6 mice, cells accumulate which have the capacity to efflux Hst in agreement with previous reports of an increase in HSC number with age in this mouse strain.
VEGF-VEGF Receptor 1 (Flt-1) Interactions Significantly Modulate Endothelial Progenitor Cell (EPC) Migration but Have Only Minor Effects on the Migration of Differentiated Endothelial Cells.
Authors: Li Bin , Teleron Amylynn A. , Lin Charles , Young Pampee P.
Published: October 2019

There is a growing body of data demonstrating that vasculogenesis, whereby bone marrow -derived circulating progenitor cells (EPCs) home to sites of neovascularization, results in significant contribution to blood vessel formation during tumor growth, ischemic injury and wound healing. Vascular Endothelial Growth Factor (VEGF) has recently been shown to augment vasculogenesis. In the current study, we examined if VEGF/VEGF Receptor (VEGFR) interactions are important for EPC recruitment. Both VEGFR1 (flt-1) and VEGFR2 (flk-1) are strongly expressed, as detected by immunofluorescent and FACS analysis, on EPCs obtained by ex vivo expansion of human peripheral blood. In a modified Boyden chamber migration assay, EPCs showed dose dependent migration to VEGF. To examine receptor specificity, EPCs were preincubated with receptor blocking anti-VEGFR1 or -VEGFR2 prior to the migration assay. Level of inhibition by VEGFR1 blocking antibody was commensurate with blocking VEGFR2. Furthermore, migration in response to a VEGFR1-specific agonist, PlGF, was comparable to that induced by VEGF and was completely ablated by preincubation with VEGFR1 blocking antibody. By contrast, differentiated endothelial cells had diminished migration in response to PlGF as compared to VEGF. Furthermore, blocking VEGFR1 only mildly disrupted VEGF-induced migration of differentiated endothelial cells in vitro. Hence, unlike differentiated endothelial cells, EPC migration in vitro was mediated by both VEGF receptors. By quantitative RT-PCR, we examined the level of VEGFR1 and VEGFR2 mRNA transcripts in EPCs versus differentiated endothelial cells. VEGFR1 transcripts in EPCs were expressed 3-fold higher than in differentiated endothelial cells. VEGFR2 and neuropilin transcript levels in EPCs, however, were lower than in differentiated endothelial cells. These results suggest that VEGF/VEGFR1 interactions are important in EPC migration in vitro. We have subcloned VEGFR1 cDNA into a retrovirus vector and have shown by western blot that we can direct increased cellular expression of VEGFR1. In further experiments, we will examine the role of VEGFR1 in human EPC recruitment using murine xenotransplant models of hindlimb ischemia and wound healing. These studies will provide valuable insight towards developing EPCs as cellular therapy to augment blood vessel formation.
Primary Nasal NK/T Cell Lymphoma: Cytology and Level of the Antiapoptotic PI9 Protein Have Prognostic Relevance.
Authors: Bossard Céline , Belhadj Karim , Reyes Felix , Martin-Garcia Nadine , Kümmer Alain , Berger Françoise , Brière Josette , Baglin Anne-Catherine , Casiraghi Odile , Lepage Eric , Gaulard Philippe ,   for the GELA
Published: October 2019

Nasal NK/T cell lymphoma is a rare disease entity characterized by a nasopharyngeal presentation, a common origin from NK cells, an association with EBV and a predilection for Asians and South Americans contrasting with its rarity in Western countries. Several studies (Chim C.S. et al., Blood 2004; Li C.C. et al., Cancer 2004) indicate that the disease has a poor outcome following conventional combined chemoradiotherapy treatments, which might be related to resistance to therapy-induced apoptosis. It has been shown (Hermine O. et al., Blood 1996; Ten Berge R. et al., Blood 2002) that high level of expression of antiapoptotic proteins by lymphoma cells is associated with poor outcome in a variety of lymphomas.

In a retrospective analysis of 37 patients treated with first-line chemotherapy (n=34) or chemoradiotherapy (n=3) according to the LNH87, LNH93 and LNH98 trials of the GELA, we have analyzed by immunohistochemistry the expression of the granzyme B-protease inhibitor 9 (PI9) regarded as an inhibitor of apoptosis, and of the active form of caspase-3 (aC-3), an effector of apoptosis. The diagnosis was based on biopsies of the upper respiratory tract showing a proliferation of neoplastic cells with a CD3+/CD5− (100%), CD56+ (90%), TIA1/GrB+ (100%) phenotype and EBV association (100%). Among the 37 patients, 73% were < 60y, 68% were male. 76% had stage I, 5% Stage II, 19% stage IV and 68% had an IPI score of 0–1. With a median follow-up of 6.3 y, 5y-event-free and overall survivals were 38% and 47%, respectively. According to cytology, tumors were subclassified into predominatly small cell (52%) and predominatly large cell (48%) categories. PI9 was scored as positive (>10% of tumor cells) in 68% of the cases, whereas 35% were assigned a high number (>10 per field at high magnification) of aC-3 positive tumor cells. Univariate analysis showed that small cell cytology and absence of PI9 expression were associated with poor outcome, but not the level of aC-3 expression nor IPI score. By multivariate analysis, cytology and PI9 downregulation were shown to independently affect event-free (P=0.01 and 0.05, respectively) and overall survival (P=0.009 and 0.006). We conclude that nasal NK/T cell lymphomas disclose cytological heterogeneity which has prognostic relevance. We also found that lack of expression of PI9 is associated with poor outcome, an unexpected finding in view of the known antiapoptotic function of this protein. Since PI9 is constitutively expressed by normal NK cells, it might be postulated that its impaired expression in tumor cells reflects a yet unknown mechanism associated with progression.
Serum-Soluble Tumor Necrosis Factor Receptor 2 (sTNF-R2) Level Determines Clinical Outcome in Patients with Aggressive Non-Hodgkin’s Lymphoma.
Authors: Tsurumi Hisashi , Goto Naoe , Takemura Masao , Hara Takeshi , Sawada Michio , Yamada Toshiki , Kasahara Senji , Kanemura Nobuhiro , Takahashi Takeshi , Oyama Masami , Tomita Eiichi , Takami Tsuyoshi , Moriwaki Hisataka
Published: October 2019

The tumor necrosis factor (TNF) plays a key role in inflammatory processes, as this cytokine is one of the earliest to be produced in such a condition, and triggers the following cytokine cascade. In addition, the TNF and their receptor system are believed to play a key role in the growth, differentiation, and/or apoptosis of the malignant cells. As for TNF receptors, the two types, the 55 kDa (p55, TNFR; TNF-R1) and the 75 kDa (p75, TNFR; TNF-R2) are simultaneously expressed on many cells at different levels. The extracellular domains of these two receptors are released from the cell membrane by cleavage of TNF-Rs as soluble TNF-Rs (sTNF-R1, sTNF-R2). Reportedly the serum TNF-Rs level rise in patients with some malignancies. The aim of the present study was to assess the prognostic significance of serum sTNF-R in aggressive non-Hodgkin’s lymphoma (NHL). Consecutive 110 previously untreated patients with aggressive NHL (diffuse large B-cell lymphoma; 94, peripheral T-cell lymphoma; 16) prospectively participated in this study between 1997 and 2002. The patients were treated with 6–8 cycles of CHOP or THP-COP regimens. To evaluate serum levels of sTNF-Rs (p55; TNF-R1, p75; TNF-R2), venous blood samples were drawn from patients immediately before the initiation of treatment. Serum sTNF-R1 and sTNF-R2 were determined using a sandwich enzyme-linked immunosorbent assay (ELISA). In healthy control subjects, the median of serum sTNF-R1 and sTNF-R2 levels were 1.2 ng/ml (range 0.3–2.9) and 4.17 ng/ml (range 1.91–8.51), respectively. High serum sTNF-R level was associated with some poor prognostic factors and low complete remission (CR) rate. Patients with high sTNF-R1(4 ng/ml and over) and sTNF-R2 (15 ng/ml and over) at onset had significantly lower survival rates (5-year: 19%, 19%) than those with low sTNF-R1 (under 4 ng/ml) and sTNF-R2 (under 15 ng/ml) (62%, 69%), respectively (p<0.0005, p<0.0001). Multivariate analysis employing sTNF-R2 and some conventional prognostic factors demonstrated that sTNF-R2 and performance status for overall survival (OS) and sTNF-R2, sIL-2R, and LDH for event free survival (EFS) were significantly poor prognostic factors. As for TNFa, a serum TNFa level is not related with sTNF-R1 or sTNF-R2 level in aggressive NHL. In addition, serum TNFa level is not associated with OS and EFS. In conclusion, serum sTNF-R2 might be a significant prognostic factor for aggressive NHL and a useful tool for selecting the appropriate therapeutic strategy in the treatment of aggressive NHL. The most reliable prognostic factor and the best combination of some prognostic factors for aggressive NHL should be clarified in order to assist in selecting appropriate treatment.
Cyclophosphamide Induced CD11b+ Cells Can Inhibit Anti-CD40 Monoclonal Antibody (mAb) Therapy of B-Cell Lymphoma.
Authors: Honeychurch Jamie , Glennie Martin J. , Johnson Peter W. , Illidge Tim M.
Published: October 2019

Immunotherapy with mAb is emerging as an important component of treatment for B-cell lymphoma. Furthermore, anti-tumor effects may be enhanced by combining mAb with a range of conventional cancer treatments and recently impressive results have been acheived with mAb in combination with chemotherapy. Here, we have assessed the efficacy of combining an agonistic anti-CD40 mAb with cyclophosphamide (CY). We have previously shown that large repeated doses of anti-CD40 mAb alone may stimulate cytotoxic T-cell lymphocyte (CTL) responses and provide robust protection in syngeneic murine tumors, such as BCL1 lymphoma. In this study we therefore used single smaller doses of the mAb that alone fail to provide long-term clearance thus allowing us to look for additive or synergistic effects of anti-CD40 and CY. Interestingly, the timing of CY relative to mAb administration appeared critical to therapeutic outcome. Cohorts given CY 7 days (d7 CY) prior to anti-CD40 show reduced survival compared to those receiving mAb alone. In contrast treatment with CY given 1 day before, or on the same day as mAb resulted in greatly increased tumor free survival. In vivo tracking experiments revealed that there is a potent CTL response that clears tumor around 5 days after anti-CD40 treatment. However, when CY was given d7 prior to anti-CD40 mAb, CTL expansion was reduced compared with other treatments, and instead resulted in increased numbers of splenic CD11b+ cells. These CD11b+ cells have an immature myeloid precursor phenotype and were able to produce nitric oxide (NO). Co-culture of CD11b+ cells with murine splenocytes resulted in greatly reduced cytotoxic CTL proliferation, but no apoptosis, which was reversed in the presence of NO inhibitors. Similarly, splenic CTL proliferation in vivo is inhibited by this increased population of CD11b+ cells. Importantly adoptive transfer of CY-induced CD11b+ into non-CY-treated mice inhibited anti-CD40 mAb tumor protection in BCL1 lymphoma. This data indicate that combining CY with immunotherapy can enhance therapeutic responses, but the timing of CY relative to immunotherapy is critical to the therapeutic outcome.
Antagonist Anti-CD40 Monoclonal Antibody, CHIR-12.12, Inhibits Growth of a Rituximab-Resistant NHL Xenograft Model and Achieves Synergistic Activity When Combined with Ineffective Rituximab.
Authors: Long Li , Tong Xia , Patawaran Montesa , Aukerman Lea , Klinger Julie , Jallal Bahija , Luqman Mohammad
Published: October 2019

CD40 and CD20 are expressed in several B-cell malignancies and represent attractive targets for therapeutic intervention. The anti-CD20 monoclonal antibody, rituximab, is an approved drug for the treatment of non-Hodgkin’s lymphomas. however, the existence of patients with rituximab-resistant disease limits its clinical utility. We have previously reported that the novel, highly potent, fully human antagonistic anti-CD40 monoclonal antibody, CHIR-12.12, generated from XenoMouse® mice (Abgenix, Inc) has greater anti-tumor activity than rituximab in both rituximab-responsive and rituximab-resistant human NHL models. In this study, we evaluated the potential therapeutic application of combining CHIR-12.12 and rituximab for the treatment of NHL. Namalwa is a Burkitt’s lymphoma cell line that gives rise to aggressive rituximab-resistant tumors when implanted in nude mice. Direct treatment of these tumor cells with CHIR-12.12 or rituximab in culture does not affect cell growth when compared to treatment with an isotype control antibody. Although Namalwa cells express more CD20 than CD40 (average of 10,059 CD20 and 3,138 CD40 molecules per cell respectively, P=0.05), when tested for in vitro ADCC killing using human NK cells as effectors, CHIR-12.12 mediated stronger target cell lysis than rituximab (31.43% vs. 14.15%, P<0.0001). Adding CHIR-12.12 and rituximab together did not enhance the in vitro ADCC killing. When CHIR-12.12 and rituximab were tested in a subcutaneous Namalwa xenograft model, CHIR-12.12 alone caused 60% tumor growth inhibition (P=0.028) whereas rituximab alone at 10 and 20 mg/kg did not inhibit Namalwa tumor growth. When tumor-bearing mice were administered rituximab at 10 mg/kg plus CHIR-12.12 at 5 or 10 mg/kg, synergistic anti-tumor activity was observed in a CHIR-12.12 dose-dependent manner. The mean tumor volume reduction in combination groups is 77% with CHIR-12.12 at 5 mg/kg (P=0.0037) and 83% with CHIR-12.12 at 10 mg/kg (P=0.0018), respectively. The potential interaction between CD40 and CD20 molecules was evaluated in vitro by treating the tumor cells with CHIR-12.12 and assessing the change in CD20 expression and vice versa. The result showed no augmentation of one antigen expression by treating the tumor cells with the other antibody. The mechanism of anti-tumor synergy observed in this combination is under evaluation. Taken together, these data suggest that the combination therapy with anti-CD40 CHIR-12.12 and rituximab has the potential to improve patient outcome in B-cell malignancies co-expressing CD20 and CD40 antigens and support the further development of CHIR-12.12 antibody for treatment of B-cell malignancies.
The Geldanamycin Analogue Heat Shock Protein 90 (HSP90) Inhibitor 17-AAG in Lymphomas: Molecular Mechanisms of 17-AAG-Induced Cell Death in Hodgkin Disease and Non- Hodgkin Lymphoma Cell Lines.
Authors: Georgakis Georgios V. , Li Yang , Younes Anas
Published: October 2019

Heat Shock Proteins (HSP) are stress-induced proteins that act as molecular chaperones in all benign and malignant cells. HSPs are important for maintaining the function of several client proteins involved in transcriptional regulation, signal transduction, and cell-cycle control. HSP90 has been recently identified as a potential therapeutic target in several types of tumors. However, the effect HSP90 inhibition in Hodgkin disease (HD) and non-Hodgkin lymphomas (NHL) remains unknown. The geldanamycin analogue 17-allylamino-17-demethoxy-geldanamycin (17-AAG) is a small molecule inhibitor of HSP90, which interferes with ATP-dependent chaperones activity, with subsequent degredation of client proteins by the proteasome. We studied the activity of 17-AAG in a panel of cell lines of HD (HD-MyZ, HD-LM2, L-428, KM-H2) and NHL (SUDHL1, and Jurkat) origin. HSP90 was expressed in all HD cell lines but not in normal peripheral blood lymphocytes of healthy donors. 17-AAG demonstrated a significant antiproliferative effect in a dose (01 micromolar - 5 micromolar) and time (24 hours to 72 hours) dependent manner in all cells lines, but was most prominant in the HD-LM2 cells. In all HD-derived cell lines, 17-AAG had more than 50% inhibitory effect over control cells. This effect was most prominant after 72 hours of incubation. Subsequently, we examined the molecular mechanisms responsible for this antiproliferative effect. Both cell cycle arrest and induction of apoptosis were observed. In the HD-LM2 cell line, 17-AAG predominantly induced a G0/G1 cell cycle phase arrest, while in the L-428 cell line there was a G2/M phase arrest (as determined by propidium iodide staining and flow-cytometric analysis). This effect was associated with alterations in MDM2 and cyclin D1 proteins. Cell cycle arrest was followed by apoptosis as determined by Annexin-V binding assay, which was predominantly mediated by caspase 9 activation. 17-AAG also reduced the cellular content of Bcl-2, cFLIP, and AKT, but had no significant effect on XIAP or cIAP2. Furthermore, 17-AAG decreased the total ERK1/2 which has been shown to play an important role in HRS cell survival. 17-AAG-induced apoptosis was partially reversed by the pancaspase inhibitor ZVAD-FMK. Preliminary data suggested that 17-AAG did not enhance the effect of doxoorubicin or gemcitabine chemotherapy. These data suggest a first proof-of-principle that inhibition of HSP90 might be of therapeutic value in HD and NHL.
Primary Mediastinal Large B-Cell Lymphoma: Long Term Results with an Alternating Chemotherapy Regimen Based on High-Dose Methotrexate.
Authors: Fietz Thomas J. , Fiedler F. , Franke Astrid , Freund Mathias , Niederwieser Dietger W. , Thiel Eckhard , Knauf Wolfgang U. ,   East German Study Group on Hematology and Oncology
Published: October 2019

Primary mediastinal large B-cell lymphomas (MLCL) differ from other diffuse large cell lymphomas: they convey a unique gene expression profile reminding of Hodgkin’s disease, are characterized by an occlusive infiltration pattern (e.g. with vena cava superior syndrome due to the concomitant sclerosis) and treatment with CHOP-based chemotherapy regimen has been disappointing. A dose intensification improves long-term results, but no standard therapy has been established so far. We investigated the use of a high-dose methotrexate based alternating chemotherapy regimen (B-ALL protocol of the GM-ALL) first as a single-center, later as a multicenter trial in 43 patients; depending on the extend of the disease mediastinal radiotherapy and/or high-dose chemotherapy with stem cell rescue were applied additionally.

15 patients have been included in our pilot single-center study (median age 31.5 years). 11/15 (73%) showed infiltration of either esophagus, pleura, lung or V.cava superior syndrome, 6/15 (40%) had pericardial effusion. In this cohort 14/15 (93%) achieved a remission (10/15 CR, 4/15 PR), which is long lasting up to 15 years as observed for the first CR patients.

28 patients have been included into the recent multicenter trial. Here the rate of evaluable remissions after 6 cycles of chemotherapy was identical with 95% (18/19) complete or partial remissions. However, 2 patients died after the first cycle of chemotherapy following septic complications. In 136 chemotherapy cycles the rates of a grade 3 or 4 toxicity were 21% (mucositis), 41% (neutropenia), 30% (thrombocytopenia) and 10% (neutropenic fever).

A methotrexate based alternating chemotherapy regimen leads to excellent remission rates in patients with primary mediastinal large B-cell lymphoma. With regard to the patients included in our single-center trial with a long follow-up these remissions are reliable and long-lasting. Nevertheless, early death following treatment initiation does occur and requires close monitoring and treatment in specialized institutions.
Treatment Outcome of Aggressive Lymphoma with CyclOBEAP (CHOP-Like + Etoposide and Bleomycin): Results of Multicenter Trial.
Authors: Okamoto Masataka , Niitsu Nozomi , Aoki Sadao , Kuraishi Yasunobu , Okumura Hirokazu , Hirano Masami
Published: October 2019

Introduction: To improve treatment outcome in aggressive lymphoma, a CHOP-like regimen with added etoposide and bleomycin was studied.

Patients and Methods: Between April 1997 and March 2003, 126 consecutive patients (pts) with aggressive lymphoma of low-intermediate to high risk disease according to the age adjusted International Prognostic Index (IPI), age<61 years, and performance status(ECOG)<3 were treated with CyclOBEAP. MCL and ATL/L were excluded from this study. CyclOBEAP was a weekly protocol for a total period of 12 weeks. It consisted of doxorubicin 50mg/m2 given every 2 weeks in combination with either cyclophosphamide 1,000mg/m2 (weeks 1, 5, 9) or etoposide 70mg/m2 qd x4 (weeks 3, 7, 11). During the alternative weeks non-myelosuppressive vincristine 1.4mg/m2 (max. 2.0mg/body) was given either with bleomycin 10mg/m2 (weeks 2, 6, 10) or alone (weeks 4,8,12). Prednisolone 40mg/m2 was administered daily for 14 days during weeks 1–2, 5–6, and 9–10. Protocol dose intensity relative to that of CHOP was 1.5 for DXR, VCR and 1.0 for CPA. G-CSF was prophylactically used. Pts who had had a bulky disease at presentation received 40Gy of local irradiation after chemotherapy.

Results: One hundred twenty-six pts were enrolled and 5 were excluded from analysis; 3 due to change of diagnosis ( ATL/L, Hodgkin lymphoma, germ cell tumor), 1 due to PS4 and the other due to drop out. In 121 eligible pts (DLBCL 94, PTCL 11, ALCL 7, nasal NK/T 4, others 5), 7 pts did not complete the full course of the treatment because of pneumonia (3), liver damage (1), intrathoracic hemorrhage (1), and PD response at 8th week (2). Relative given dose intensity was 0.94 for DXR, 0.95 for CPA, 0.86 for VCR, 0.94 for VP16, and 0.96 for BLM. All pts were evaluated. CR (including cCR) was obtained in 106 (88%), PR in 11 and NC/PD in 4. No treatment related death was observed. With a median follow-up of 56 months, 5yr overall survival (OS) rate is 72% and progression-free survival (PFS) rate is 62%. The 5yr OS and PFS within the subgroups defined by IPI and in pts with DLBCL and PTCL are not significantly different (data shown in the table). WHO grade 4 neutropenia was observed in 91 pts and thrombocytepenia in 13 pts. 66 pts required 2 or more units of red cell transfusions (2 – 66 units). Liver damage of grade 3 to 4 was seen in 3 pts.

Conclusions: Addition of etoposide and bleomycin to CHOP drugs may enhance the effect of CHOP for aggressive lymphoma, encouraging comparative study between the two treatments.

n CR (%) 5yr OS 5yr PFS *OS:Log-Rank 0.578; Wilcoxson 0.616 *PFS:Log-Rank 0.552; Wilcoxson 0.573 #OS:Log-Rank 0.049; Wilcoxson 0.059 #PFS:Log-Rank 0.122; Wilcoxson 0.146 All 121 106 ( 88) 72 % 62 % IPI: L-I* 41 37 ( 90) 69 65 IPI: H-I* 62 56 ( 90) 71 55 IPI: H* 18 13 ( 72) 82 43 DLBCL# 94 82 ( 87) 70 58 PTCL# 11 11 (100) 100 82
Combination Therapy with CC-5013 (Lenalidomide; Revlimid™) Plus Dexamethasone (Rev/Dex) for Newly Diagnosed Myeloma (MM).
Authors: Rajkumar S. Vincent , Hayman Suzanne R. , Lacy Martha Q. , Dispenzieri Angela , Hoering Antje , Geyer Susan M. , Zeldenrust Steven R. , Greipp Philip R. , Fonseca Rafael , Lust John A. , Russell Stephen J. , Kyle Robert A. , Witzig Thomas E. , Gertz Morie A.
Published: October 2019

Background: Increasingly thalidomide (Thal) plus dexamethasone (Dex) is being used as initial therapy for multiple myeloma (MM), but there is a need to minimize non-hematologic toxicity with this regimen. CC-5013 (lenalidomide; Revlimid™) is a more potent analog of thalidomide with significantly fewer non-hematologic toxicities that has shown promising results in relapsed refractory myeloma. We report the initial results of the first phase II trial using the combination of CC-5013 plus Dex (Rev/Dex) as initial therapy for newly diagnosed MM.

Methods: The trial is designed to accrue 31 eligible patients; 13 patients (pts) (11 male and 2 female) were analyzed in this interim report. Patients were enrolled between February 2004 and July 2004. CC-5013 was given orally at a dose of 25 mg daily on days 1–21 of a 28-day cycle. Dex was given orally at a dose of 40 mg daily on days 1–4, 9–12, 17–20 of each cycle. Patients also received an aspirin once daily as thrombosis prophylaxis. Response was defined as a decrease in serum monoclonal (M) protein by 50% or higher and a decrease in urine M protein by at least 90% or to a level less than 200 mg/24 hours. Responses were assessed on an intent to treat basis.

Results: The median age was 61 years (range, 32–78). 8 patients (62%) had Stage III myeloma. 11 of the 13 patients achieved an objective response yielding a response rate of 85% within 1–2 months of therapy. So far 6 patients have experienced grade 3 adverse events. These include one episode each of CD4-count < 200/mm3, anemia, neutropenia, increased liver enzymes, muscle weakness, agitation, hyperglycemia, cardiac arrhythmia, pneumonitis, and colonic perforation (underlying diverticulitis and dexamethasone suspected). No deep vein thrombosis or grade 4 or higher adverse events have been observed so far.

Conclusions: Rev/Dex appears active and well tolerated in the treatment of newly diagnosed MM and is a potential alternative to Thal/Dex. However, these results are preliminary and responses are still being evaluated and need to be confirmed in the final analysis of this trial. A large randomized trial using Rev/Dex as initial therapy for MM is expected to be activated by the Eastern Cooperative Oncology Group later this year.
Thrombopoietin Protects Against Cardiotoxicity Induced by Doxorubicin.
Authors: Sung Rita Y.T. , Huang Wei Zhe , Yang Mo , Pong Nga Hin , Lee Shuk Man , Chan Wood Yee , Wong Yeuk Oi , Xiao Da Wei , To Man Yin , Li Karen K.H.
Published: October 2019

Doxorubicin (DOX), an anthracycline drug, is the key component of various chemotherapy regimens. However, it exerts severe cardiotoxicity including cardiomyopathy and congestive heart failure. Thrombopoietin (TPO) is an established cytokine for promoting early hematopoietic progenitor cells, the megakaryocytic/platelet lineage and angiogenesis. In this study, we investigated effects of TPO on the protection against cardiotoxicity induced by DOX in a mouse model. Male Balb/c mice at 10 weeks of age were randomly divided into 3 groups (each N=6): Normal control (saline), DOX (20mg/kg, single dose i.p., Day 0) and TPO+DOX treatment (12.5μg/kg TPO at Days −1, +1 and +3). Transthoracic echocardiography (Sonos 7500) was performed using a high frequency transducer (15L6) at baseline (Day −1) and Day +5 on animals maintained at a conscious state. Our results demonstrated that DOX exerted cardiotoxicity in DOX-treated animals, indicated by significant reductions in heart weight, heart rate and fraction shortening (Table). Treatment with TPO significantly increased these heart function parameters. Effects of TPO were confirmed by the morphology of heart tissues, which demonstrated extensive cytoplasmic vacuolization (mean±SD = 20.0±3.30%) and myofibrillar loss (42.0±5.90%) in DOX-treated animals. These damages were significantly reduced in TPO+DOC-treated mice (8.00±1.60% and 21.0±3.30% respectively, p<0.05). The possible direct effects of TPO on rat cardiomyocytes were further investigated by in vitro studies on cell line and primary cells. The MTT assay on the H9C2 cell line demonstrated that DOX (5μg/ml, 24h) significantly reduced cell viability by 26.2±1.09% (n=4, p<0.01). These cells responded to TPO at a dose-dependent manner and cell viability was recovered to 85.6±4.43% (100ng/ml TPO, p<0.01). DOX (0.58μg/ml) inhibited primary neonatal cardiomyocytes in culture (48h) by reducing their beating rates to 9.25±11.1% of those recorded before DOX treatment (n=8, p<0.01). TPO and the cardioprotective drug dexrazoxane (5.8μg/ml) increased the beating rates of these cardiomyocytes to 47.8±12.7% and 34.5±11.5%, respectively (p<0.01). We also detected the expression of TPO receptors (c-mpl) by RT-PCR in mouse heart tissues. Our study demonstrated that TPO has cardioprotective effects against DOX toxicity and could be further developed for clinical applications.

Effects of TPO on Heart Weight and Functions of DOX-Treated Mice

Heart Weight (mg) Heart Rate (beats/min) Fraction Shortening (%) Mean±S.D.; * p<0.01 (DOX vs Control); # p<0.05 (DOX vs TPO+DOX at Day+5); ** p<0.05 (Day-1 vs Day+5) Control (Day-1) - 551±41.0 57.0±4.30 Control (Day+5) 118±15.3 581±40.0 60.7±2.80 DOX (Day-1) - 605±37.3 58.7±1.53 DOX (Day+5) 79.2±8.17* 410±45.2** 39.5±4.93** TPO+DOX (Day-1) - 575±40.0 60.2±3.55 TPO+DOX (Day+5) 92.7±6.94# 515±67.0# 56.3±7.66#
Myeloablative Treosulfan as Preparative Regimen for Allogeneic Haematopoietic Stem Cell Transplantation: A Single-Centre Experience.
Authors: Trenschel Rudolf , Ditschkowski Markus , Elmaagacli Ahmet , Steckel Nina K. , Hlinka Michal , Peceny Rudolf , Ottinger Helmut , Koldehoff Michael , Biersack Harry , Flasshove Michael , Beelen Dietrich W.
Published: October 2019

Treosulfan (TREO), a water-soluble bifunctional alkylating agent, has demonstrated strong immunosuppressive and antileukemic activity as well as profound stem cell toxicity in animal studies. Due to the advantageous clinical toxicity profile lacking significant non-hematologic organ toxicities, high-dose TREO in combination with cyclophosphamide (CY) has recently been evaluated in patients (pts) with an increased risk for organ toxicities precluding standard myeloablative conditioning regimens before allogeneic stem cell transplantation (SCT). Between 8/00 and 10/03, we treated 52 patients (pts) not eligible for conventional therapy with TREO in order to reduce toxicity in a myeloablative setting. Diagnoses were AML (n=14), ALL (n=11), MM (n=8), NHL (n=7), MDS (n=5), CML (n=4) and aplastic syndromes (n=3). 18 patients were grafted in early disease (1st or 2nd complete remission, chronic phase, or incipient first relapse (BM blasts < 10%). The remaining pts were classified as having advanced disease. Donors were identical siblings (n=24), non-identical family members (n=l), matched unrelated (n=14) or mismatched unrelated (n=13) donors. Conditioning regimen consisted of TREO 36g/qm (n=19) or 42g/qm (n=28) and CY 120mg/kg BW, 5 pts received TREO 42g/qm and fludarabine 150mg/qm. GvHD prophylaxis consisted of CSA alone (n=l) or in combination with short course MTX (n=25), alemtuzumab (n=22) or ATG (n=4). ANC > 500/μl and platelets > 20000/μl were reached at day 15 and 16 respectively. Acute GvHD grade II - IV occurred in 31% of pts and chronic GvHD in 60% of pts. Overall (OS) and disease free survival (DFS) were closely related to disease status. OS and DFS was 93% and 82,9% after a median of 18 months (range 0,9–38,5 months) for pts with early disease. In advanced disease the OS was 57,4% and the DFS 47,9% after a median of 4,8 months (range 0,3 – 22,9 months), respectively. In early disease, a single patient died of invasive aspergillosis associated with grade IV aGvHD. Another patient developed a relapse of CML which was successfully treated with DLI. Clinical relevant adverse events occurred in patients with advanced disease: MOF (n=7), VOD (n=2), infectious problems associated to GvHD grades II – IV (n=4), and pulmonary embolism (n=l). TREO as part of a myeloablative regimen seems to be effective and safe even in pts not eligible for conventional myeloablative therapy.
Pleitrophin Is Highly Expressed by Myeloma Cells, Elevated in the Serum of Myeloma Patients, and Is a New Autocrine Growth Factor for This B Cell Malignancy.
Authors: Chen Haiming , Zhu Daocheng , Campbell Richard A. , James Christine Pan , Li Sonia Guangxu , Santos Janice , Wang Cathy S. , Bonavida Benjamin , Berenson James R.
Published: October 2019

Pleitrophin (PTN) was originally described as a development regulatory cytokine. This protein regulates the growth of neuroectodermal and mesodermal cell lineages during early embryogenesis but becomes down-regulated during late phase of embryogenesis and shows a very restricted expression pattern in adult neural system. This protein has been recently shown to both induce angiogenesis and bind to syndecan. Thus, we determined whether this protein may be found in tumor cells from myeloma patients and its potential to influence their growth. First, we measured serum levels of PTN in myeloma (n=115) and age-matched controls (n=50) using an ELISA-based technique. PTN levels in the serum of myeloma patients were markedly elevated compared to the normal control group (P<0.02). The serum concentration of PTN in MM patients averaged 28 pg (range, 8 to 110 pg). In contrast, the control serum levels averaged only 12 pg (range, 0 to 20 pg. Interestingly, the PTN concentration in two patients with plasma leukemia was much higher than other multiple myeloma patients. Next, we analyzed the expression of PTN using RT-PCR on RNA from myeloma cell lines (RPMI8226, U266), and multiple myeloma patients’ and normal control bone marrow aspirates. Results showed that the PTN mRNA was strongly expressed in multiple myeloma cell lines, myeloma bone marrow samples but not in the normal control bone marrow specimens.

In order to determine whether PTN stimulates multiple myeloma growth, we further cloned a whole PTN sense or anti-sense sequencing DNA into the multiple myeloma cell lines RPMI8226 and U266. Cells transduced with sense PTN showed markedly increased proliferation compared to cells transduced with antisense or vector alone. The role of PTN in hematological malignancies has not been previously defined. Due to the restricted expression pattern of PTN in adults, PTN is suggested as a potential new target for treatment of multiple myeloma. Further investigations are defining the prognostic value of PTN serum levels and the mechanism by which this cytokine drives myeloma growth.
MTOR Inhibitors Activate the AKT Kinase in Multiple Myeloma Cells by Upregulating the IGF-1/IRS-1/PI-3 Kinase Cascade.
Authors: Shi Yijiang , Yan HuaJun , Frost Patrick , Hoang Bao , Gera Joseph , Lichtenstein Alan
Published: October 2019

MTOR inhibitors, such as rapamycin and CCI-779, have shown pre-clinical potential as therapy for multiple myeloma (MM). By inhibiting expression of cell cycle proteins, these agents induce G1 arrest. However, by also inhibiting an mTOR-dependent phosphorylation of insulin receptor substrate-1 (IRS-1), they may alter its subcellular localization and/or prevent its degradation which could enhance IGF-1 signaling and downstream PI3-kinase/AKT activation. This may be a particular problem in MM where IGF-1-induced activation of AKT is an important anti-apoptotic cascade. We, thus, studied PI3-kinase/AKT activation in MM cells treated with mTOR inhibitors. Rapamycin enhanced basal AKT activity, AKT phosphorylation and PI3-kinase activity in MM cell lines. Both PTEN-null as well as PTEN-wild type myeloma lines were similarly affected. Rapamycin also significantly prolonged activation of AKT induced by exogenous IGF-1. CCI-779, used in a xenograft model, also resulted in MM cell AKT activation in vivo. Blockade of IGF-1 receptor function prevented rapamycin’s activation of AKT. Furthermore, rapamycin prevented serine phosphorylation of IRS-1 and IRS-1 degradation. Though similarly blocking IRS-1 degradation, proteasome inhibitors did not activate MM cell AKT. Although rapamycin sensitized MM cells for dexamethasone-induced apoptosis, it protected against PS-341-induced apoptosis. Thus, mTOR inhibitors activate PI3-K/AKT in MM cells and activation depends on basal IGF-1/IGF-R signaling. As activated AKT may protect against apoptosis, future use of mTOR inhibitors in myeloma patients will have to carefully consider the types of anti-myeloma agents used in combination.