bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Analysis of Coagulation System Proteins Distribution in Breast Cancer Primary Lesions and Lymph Node Metastases.
Authors: Wojtukiewicz Marek , Sierko Ewa , Sawicki Zbigniew , Zimnoch Lech , Kisiel Walter
Published: October 2019

Breast cancer is the most frequent cause of morbidity and mortality due to cancer in women. Thromboembolic episodes are relatively common complication during the course of the disease. It was widely documented, that components of blood coagulation play a role in tumor progression and metastasis formation. Presence of hemostatic proteins was observed in primary lesions of different malignant tumors. However, information on expression and role of coagulation system components in regional lymph node metastases is scanty. The purpose of the study was to assess and compare the distribution of selected coagulation proteins - in primary versus metastatic lesions of breast cancer. Specimens of primary tumor of different degree of malignancy and metastatic lesions localized in regional lymph nodes were obtained during surgical treatment of previously untreated twenty five breast cancer patients and fixed in formalin. Immunohistochemical procedure was employed using monoclonal and polyclonal antibodies against tissue factor (TF), tissue factor pathway inhibitor (TFPI), factor IX, factor X, fibrinogen and fibrin. A strong expression of TF was demonstrated in cancer cells of both primary tumors and lymph node metastases. Coagulation factors IX and X were also present in primary and metastatic breast cancer lesions. However, the expression of the proteins was weaker in regional lymph node metastases than in primary tumor. Strong staining for fibrinogen was detected in the stroma of primary breast cancer, particularly at the area adjacent to the cancer foci. Stroma of metastatic lesions was characterized by much weaker staining for fibrinogen. Strong expression of fibrin and TFPI was demonstrated in cancer cells of primary tumor while much weaker expression of the antigens was visualized in cancer cells localized in lymph node metastatic lesions. Furthermore, the presence of all examined proteins was observed in small blood vessel walls and in macrophages infiltrating both primary tumor tissue as well as metastatic lesions. The results of the study indicate extravascular activation of blood coagulation in loco in breast cancer, both in the primary tumor and in the regional lymph node metastatic lesions. The presence of coagulation system proteins in cancer tissue (primary tumor and metastatic foci) suggests that they may play a role in the biology of breast cancer growth as well as they may modulate breast cancer metastatic dissemination.
Effects of “Primary Prophylaxis” and “On-Demand” Therapy on the Clinical Expression of Severe Hemophilia A in Children - Results of a Multicenter Study.
Authors: Schobess Rosemarie , Kurnik Karin , Kreuz Wolfhart , Friedrichs Frauke , Krumpel Anne , Bidlingmaier Christoph , Ettingshausen Carmen Escuriola , Nowak-Gottl Ulrike
Published: October 2019

Background: Patients with severe hemophilia A (HA) can either be treated by regular FVIII infusions twice or thrice per week (prophylaxis), or only in case of bleeding episodes (on-demand). Whereas prophylaxis reduces the number of bleeding episodes and may therefore prevent the development of hemophilic arthropathy, recommendations regarding age and dose at start of prophylactic regimens are still a matter of debate. The present cohort study was performed to investigate the role of “primary prophylaxis” versus “on-demand” therapy in HA children. The outcome variable was imaging-proven hemophilic joint damage.

Methods: 42 children were initially treated with primary prophylaxis following the first bleeding episode, and were frequency-matched (year of birth, catchment area) to 67 patients receiving “on-demand” therapy with an early switch to “secondary prophylaxis”.

Results: In multivariate analysis adjusted for the HA mutation type and the presence or absence of thrombophilia, the Pettersson score investigated at a median age of 12.5 years in joints with at least one documented bleeding episode was no significantly different between the two patient groups (p=0.944), and no statistically significant differences were found in patients with target joints (p=0.3), or in children in which synovitis had occurred (p=0.77). Imaging results obtained showed a substantial agreement (87.14%) beyond that expected by chance alone (42.4%) between local and central readers in the patients tested (kappa=0.77; Z= 17.27; p < 0.001).

Conclusion: In cases with severe HA where primary prophylaxis is impossible, the procedure to switch from “on-demand” to early secondary prophylaxis can be achieved in the majority of young children affected. In addition, the needs of parents around the time of diagnosis of severe HA could be better addressed.
In Vitro and In Vivo Characterization of Full-Length rFVIII Modified with PEG Via Coupling to Primary Amino Groups.
Authors: Turecek Peter L. , Siekmann Jürgen , Gritsch Herbert , Váradi Katalin , Ahmad Rafi-Uddin , Muchitsch Eva-Maria , Ehrlich Hartmut , Schwarz Hans Peter
Published: October 2019

Chemical modification of recombinant therapeutic proteins with PEG has been shown to enhance the biological half-life. Here we assess the effect of PEGylation on FVIII. Full-length rFVIII bulk drug substance from protein-free fermentation (Advate process, Baxter) was conditioned into a buffer suitable for coupling to polyethylene glycol succinimidyl succinate (linear PEG, 5 kDa PEG chain length). PEG was covalently bound by amine coupling preferentially to lysine residues of FVIII at neutral pH. PEG was removed by ion-exchange chromatography and the PEG-FVIII derivative was concentrated by ultra-diafiltration. The conjugates thus obtained retained about 30–40% of the activity of non-modified rFVIII. The specific activity decreased with the amount of PEG linked to the FVIII molecule. In SDS-PAGE and immunoblot studies PEGylated rFVIII showed a band pattern similar to unmodified FVIII with full-length, heavy chain fragments of 180 kDa and 120 kDa and the light chain fragment of 80 kDa. PEGylation also occurred to a high extent in the B domain of FVIII. All bands appeared broadened due to the attachment of polymeric PEG. The maintenance of functionality of FVIII was demonstrated by its potential to be activated and inactivated by thrombin. In the assay PEGylated and unmodified FVIII were incubated with 1 nM thrombin. Sub-samples were drawn at intervals up to 40 minutes and added to a mixture of FIXa, FX, phospholipid vesicles and Ca2+ containing a thrombin inhibitor. After 3 minutes incubation at 37°C the amount of activated FX (FXa) was measured using a FXa-specific chromogenic substrate. Unmodified rFVIII showed a typical picture of an immediate increase in FXa activity and a subsequent decline with no further FXa generation after 15 minutes. PEGylated rFVIII was activated to the same extent as unmodified FVIII but the decay in FXa generation was slower and did not reach the zero level, even 40 minutes after incubation. The formation of the typical thrombin cleavage fragments, with unmodified as well as PEGylated rFVIII, was demonstrated in a Western blot analysis. The slower inactivation by thrombin was also seen there. The pharmacokinetic properties of PEGylated rFVIII compared with rFVIII were investigated in hemophilia A knock-out mice. Both preparations were applied at a dose of 200 IU rFVIII/kg and groups of mice (n=5) were exsanguinated at several time points up to 24 hours. Terminal half-life for PEGylated rFVIII was calculated at 4.9 hours compared with 1.9 hours for unmodified rFVIII in hemophilia A knock-out mice. AUC was approximately doubled. These results indicate that rFVIII can be biochemically modified with PEG whilst at least partly retaining its major functions, but at the same time prolonging its survival in the circulation of hemophilic mice.
Prevalence Estimates of Adult Chronic Idiopathic Thrombocytopenic Purpura (ITP) in the United States.
Authors: Feudjo-Tepie Maurilee A. , Robinson Noah J. , Bennett Dimitri
Published: October 2019

ITP is a disease caused by inadequate platelet production as well as increased platelet destruction. Literature on the epidemiology of ITP is currently sparse and the disease remains poorly described. Based on previous reviews, the overall incidence of ITP among adults ranges from 1.6 to 3.0 per 100,000 person years of observation (PYO); prevalence estimates range from 2.1 to approximately 36.4 per 100,000 persons (Neylon AJ et al., Br J Haematol. 2003; Satia J et al., Eur Hematol Assoc. [poster presentation] 2006; Deitz AC et al., Blood. 2006). In this study, the objective was to estimate the prevalence of adult chronic ITP (in 2005) stratified by age and gender using a large US claims database (Integrated Healthcare Information Services, IHCIS). IHCIS comprises over 70 million patients from 45 health plans. Adult chronic ITP cases were defined as patients with at least two diagnoses (ICD-9 code 287.3) for primary thrombocytopenia separated by at least 6 months between 2002 and 2006, and age 18 or older in 2004. Although ICD-9 code 287.3 is not exclusive to ITP, its sensitivity and specificity are likely to be high (Segal et al., Am J Hem. 2004). A patient’s first identified 287.3 ICD-9 code over the period 2002–2006 had to be in 2004 or earlier and the last one in 2004 or later. Patients had to have continuous enrollment throughout the year 2004. We estimated the 2004 prevalence as the total number of identified chronic ITP cases divided by the total population in the database in 2004 stratified by age group and gender. Estimates of adult cases for the US population were obtained by multiplying the 2004 prevalence estimates by the 2005 adult US census population estimates. The estimated adult prevalence was 23.6 [95% CI 23.4 – 23.8] per 100,000 persons, translating to approximately 52,700 (95% CI 52,200 – 53,100) adult chronic ITP cases in the US. Females had higher prevalence than males (28.1 vs 18.8 per 100,000). In addition, prevalence increased with age (from 17.0 in the age group 18–49 to 36.2 in the age group 65 and older). These results are broadly similar to previously published data and help to better understand the epidemiology of chronic ITP. While the tight CIs predominantly reflect the large data set rather than the precision of the estimates, we believe these data are among the most robust estimates of ITP prevalence and are the first to specifically estimate adult chronic ITP prevalence by age and gender in the US.
Identification of New Alloantigen-Reactive CD8+ Cytotoxic and Suppressor T Cell Subpopulations.
Authors: Bohana-Kashtan Osnat , Levitsky Hyam , Civin Curt I.
Published: October 2019

We sought to develop a better understanding of the T cells involved in the human allogeneic immune response, in order to eventually engineer a donor graft with reduced GVHD-mediating potential, without ablating general immune competence. Prior studies reported that all the activated CD4+ T cells responding to a specific antigen challenge reside within the CD4high population expressing high levels of membrane CD4. We identified a new population of activated CD8+ T cells that developed during an in vitro allogeneic immune response, along with the allo-activated CD4high T cell population. Analogous to activated CD4+ T cells, this new T cell population was distinguished by up-regulated CD8 (and CD38) expression (CD8highCD38+). In accordance with Martins et al. (Blood 2004, 104:3429), we found that the depletion of the CD4highCD38+ population resulted in reduced 2o response to the original 2nd party stimulators. In contrast, depletion of the CD8highCD38+ population resulted in an increased 2o response to 2nd party cells, with no change in the response to 3rd party or CMV antigens. Elevated numbers of CD8highCD38+ T cells potently reduced the 1o and 2o responses to 2nd party, but not to 3rd party cells or CMV antigens. The complementary, non-activated CD8normalCD38− T cell population had no inhibitory effect. Importantly, we found that

CD8highCD38+ T cells mediated both a specific cytotoxic response (that could be inhibited by the pan-caspase inhibitor, Z-VAD), and a specific suppressive response toward the original 2nd party stimulators (that was not affected by Z-VAD), and within this CD8highCD38+ population, there was a subpopulation of cytotoxic T cells (perforin+LAMP1+CD56+CD11b+CD11c+) and a subpopulation of non-cytotoxic T cells.

Furthermore, we found that although CD8highCD38+ T cells differentially expressed CD28, both CD8highCD38+CD28− and CD8highCD38+CD28− T cells mediated a cytotoxic as well as a suppressor T cell response toward the original 2nd party cells (different from the published suppressive function of CD8+CD28− T cells observed by Liu et al, Int Immunol 1998, 10:775). Upon separation of cytotoxic CD8highCD38+ T cells from suppressor CD8highCD38+ T cells, we will explore the GVHD potential of these 2 novel activated CD8high T cell subpopulations, in a sensitive in vivo xenograft model for GVHD using NOD/SCID/IL2Rγnull immunodeficient mice.
The Use of alpha Interferon To Augment the Graft-Versus-Leukaemia Effect of 2nd Stem Cell Transplants and Donor Lymphocyte Infusions in High Risk Paediatric Leukaemias.
Authors: Cooper Nichola , Amrolia Persis , Goulden Nick , Rao Kanchan , Veys Paul
Published: October 2019

Childhood leukaemia which is refractory to chemotherapy or which relapses early following stem cell transplantation (SCT) has a very poor prognosis, with few patients becoming long-term survivors. Establishing limited graft-versus-host disease (GVHD) in an attempt to exert a graft-versus-leukaemia (GVL) response in these patients remains the only curative option. Second SCT procedures and/or donor lymphocyte infusions (DLI) have achieved some success but the pace of acute leukaemia is often too fast and the occurrence of GVHD/GVL too unpredictable to achieve this goal. We postulated that the addition of alpha interferon (alphaIFN) might induce/augment and control GVHD/GVL in this group of patients and so improve outcome. Thirteen patients underwent initial SCT, for cALL CR2 (n=1), Philadelphia positive ALL CR1 (5), MDS/AML (2), refractory AML (2), and CML (3), with unrelated (9) or sibling donors (4). Eight patients subsequently had a frank haematological relapse and 4 patients a molecular relapse of their leukaemia at a median of 10 months (2–18 months) from 1st SCT. Six patients underwent a 2nd SCT from the same donor, all with reduced intensity conditioned regimes; all received alphaIFN when GVHD did not occur after early withdrawal of immunosuppression. One (ALL CR2) received DLI 2 months after 2nd SCT for mixed chimerism. A further six patients received DLI plus alphaIFN, 3/6 together with low dose DLI (<106/kg); 3/6 had previously received108/kg DLI alone without response. One patient with refractory AML received alphaIFN in conjunction with the initial transplant. The dose of alphaIFN was escalated from 3–5 megaunits/m2 subcutaneously 3–5 days per week as tolerated and continued in the absence of GVHD for up to one year. 3 patients (CML) received concomitant therapy with Glivec, which has now been stopped. Following therapy, 9/13 (69%) patients developed aGVHD: Grade I (1), grade II (4), grade III (4); and 7/11 evaluable patients cGVHD: limited (4), extensive (3). 8 of 13 (62%) remain well and in remission, a median of 23 months (range 12 to 100) from alphaIFN therapy. Five have died: 2 from relapse, one from lung GVHD, one from idiopathic pneumonitis and one from aspergillus infection. Two of 8 surviving patients have ongoing cGVHD requiring immunosuppressive therapy. This data shows promising results for the use of alphaIFN to augment GVL responses in patients with high-risk leukaemia not cured with standard transplant procedures with 62% disease free survival and minimal residual symptoms. Further analysis is warranted.
In-Vivo Immune Modulating Effects of the Human Chorionic Gonadotropine Hormone.
Authors: Elmaagacli Ahmet H. , Koldehoff Michael , Beelen Dietrich
Published: October 2019

Human chorionic gonadotropine (hCG) is a naturally occurring immune modulating agent, which is highly expressed during pregnancy. It is often observed that during pregnancy some autoimmune diseases such as multiple sclerosis and Crohn’s disease improves in women. Here we hypothesized that the improvement might be associated with HCG expression during pregnancy and evaluated its possible immunemodulating effects. Blood samples obtained from 34 women (median age 37, range 18–47) who underwent in-vitro fertilization (IVF) and received therefore HCG subcutaneously at three different time points (10.000 IE first, after 24 hours 5000IE, after 48 hours 5000IE) were examined prior and after HCG application. Thereafter skin transplantation was performed with HCG and without in mice. We noticed a significant increase of the number of Tr cells (CD4+CD25+) to up to 40% (p<0.04) after the first and second hCG application in the blood samples of women. A twofold increased of CTLA-4 expression after the first hCG application (p<0.049) was observed, whereas at the same time FoxP3 expression fell rapidly (p<0.006). Our analysis of Th1 (CD3+CD4+IFNγ) and Th2 (CD3+CD4+IL4+) type T cells revealed a slight shift after hCG application: the number of Th1 cells did not alter significantly, whereas the number of Th2 cells increased significantly (p<0.048). Further analyses showed that other subpopulations including B cells (CD19+), NK cells (CD16+CD56+), T helper cells (CD3+CD4+; CD3+CD4+CD45RA; CD3+CD4+CD45RO+), cytotoxic T cells (CD3+CD4+CD8+), TCRab and TCRgd positive T cells were not influenced by hCG. Also, we analyzed cytokine serum levels in women before and after each hCG application by ELISA. Hereby we found that two applications of hCG increased serum levels of both IL-8 and IL-10 significantly (p<0.013 and p<0.03, respectively) measured by ELISA assays (p<0.013), whereas the serum levels for IL-2, IL-4, TNF-α, IFN-γ, TGF- β and IL-1β (data not shown) were not significantly altered by hCG. HCG application increased the expression of anti-inflammatory interleukin-27 (IL-27) and reduced that of the inflammatory IL-17. In addition, we found increased IL-10 serum levels and increased numbers of regulatory T cells CD4+CD in peripheral blood of women after hCG application. Rejection of BALB/c skin grafts transplanted in B6 mice was significantly delayed when mice received hCG after transplantion. We conclude that hCG may be useful for the induction of immune tolerance in allogeneic transplantation.
Identification of Two Novel Homozygous HAX1 Mutations in an Autosomal Recessive Jewish and Two Unrelated Sporadic British Families with Severe Congenital Neutropenia.
Authors: Smith Bradley N. , Ancliff Phil J. , Linch David C. , Gale Rosemary E.
Published: October 2019

Severe chronic neutropenia (SCN) is a rare disorder characterized by profound neutropenia, recurrent bacterial infections and bone marrow arrest at the promyelocyte/myelocyte stage. Initially described as an autosomal recessive (AR) disease (Kostmann disease), autosomal dominant (AD), sporadic and X-linked forms have been reported. Pathogenic mutations in the neutrophil elastase ELA2 gene have been documented in both SCN and the related disorder cyclic neutropenia in a significant proportion of AD and sporadic but not AR patients, and mutations in the Wiskott-Aldrich syndrome (WAS) gene have been reported in X-linked SCN families. More recently, homozygous mutations resulting in AR disease were identified through linkage to chromosome 1p21 in Kurdish and the original Kostmann families, revealing mutations in the HAX1 gene that result in complete loss of this mitochondrial anti-apoptotic protein. In order to determine the frequency of these different mutations in SCN, we used denaturing HPLC WAVE analysis to investigate a cohort of 105 SCN kindreds seen in our institute or referred with a diagnosis of SCN of which 11 were presumed/known AD, 17 AR, 73 sporadic and 4 had X-linked disease. Overall, 25 different ELA2 mutations were detected in 31 kindreds (6 AD and 25 sporadic, 30% of the cohort). Mutations were distributed throughout the gene and were most frequent in exon 4 (42% of mutations), least frequent in exon 3 (10%). Two kindreds (2%) had WAS mutations as previously published, no new cases were identified. The remaining 72 cases were screened for all 7 HAX1 exons using mixtures of patient and control PCR products to detect homozygous substitutions. Four homozygous mutant kindreds were identified. One AR kindred of Kurdish descent had the previously reported p.Trp44X mutation. Two sisters of Jewish origin had a homozygous insertion of an extra G in a run of 5 Gs (nucleotides 121–125) in exon 2 resulting in a frameshift (p.Ser43LeufsX11); both parents were shown to be heterozygous for the mutation. Two unrelated sporadic SCN patients of Caucasian (British) origin had deletion of nucleotide G91 in exon 2 resulting in a frameshift (p.Glu31LysfsX54); to date, parents of one case have been confirmed as heterozygotes. Both frameshift mutations are predicted to result in a non-functional protein. The 4 HAX1-mutant kindreds have variable clinical severity (ranging from mild to severe) and G-CSF requirement (none to regular administration). All heterozygous parents are phenotypically normal. Our results indicate that HAX1 mutations are relatively uncommon (4% of our cohort) but not limited to patients with specific ethnic origin or apparent AR inheritance.
Cost-Effectiveness of Posaconazole vs Fluconazole in the Prevention of Invasive Fungal Infections among Patients with Graft-Versus-Host Disease (GVHD) in the US.
Authors: O’Sullivan Amy K. , Weinstein Milton C. , Pandya Ankur , Thompson David , Langston Amelia , Perfect John , Papadopoulos George
Published: October 2019

Trial data suggest that posaconazole is similar to fluconazole in preventing invasive fungal infections (IFIs) among allogeneic progenitor cell transplant recipients with graft-versus-host disease (GVHD). We estimated the cost-effectiveness of posaconazole versus fluconazole in this population in the US. A decision-analytic model was developed to estimate the average per patient treatment costs, IFIs avoided, life-years gained, and incremental cost per life-year gained of prophylaxis (2006 US$). The model extrapolates the trial results to a lifetime horizon to include long-term mortality due to GVHD. In the model, patients are assumed to receive posaconazole or fluconazole; efficacy data were obtained from the clinical trial. Long-term mortality and prophylaxis drug and IFI treatment costs were estimated from secondary sources. One-way and probabilistic sensitivity analyses were conducted. Posaconazole is associated with fewer IFIs (0.05 vs. 0.09), increased life years (7.87 vs. 7.66), and higher IFI-related costs (prophylaxis and IFI treatment) ($8,750 vs. $5,530) per patient relative to fluconazole. Costs for treatment of IFIs comprised 95% of the total cost for fluconazole and 35% for posaconazole. The incremental cost-effectiveness of posaconazole versus fluconazole is estimated to be $15,700 per life-year saved. Results are most sensitive to changes in the cost of treating an IFI and the efficacy of prophylaxis. Results from the probabilistic analysis indicate that there is an 88% probability that posaconazole is cost-effective at a $50,000 per life year saved threshold. We conclude that posaconazole is a cost-effective strategy for the prevention of IFIs in patients with GVHD.
Survivin Inhibition of p34Cdc2 Via Protection of Wee1 Correlates with Survival.
Authors: Guzman Javier Rivera , Fukuda Seiji , Pelus Louis
Published: October 2019

The inhibitor of apoptosis protein Survivin and p34Cdc2 (the cyclin-dependent kinase, Cdk1) are involved in cell cycle progression and apoptosis. Cdc2 and Survivin are tightly regulated in normal cells but deregulated in cancer. We have previously shown that Survivin regulates entry of hematopoietic stem cells into cell cycle. It has been established that Cdc2 phosphorylates Survivin at threonine 34 (T34), which is believed to be a stabilizing event necessary for normal Survivin function and anti-apoptotic activity. Activation of Cdc2 is required for its apoptotic activity and inhibition of the proapoptotic activity of Cdc2 can be achieved by phosphorylation at tyrosine 15 (Tyr15). Stable overexpression of a wild-type (wt) mouse Survivin-IRES-GFP construct in IL-3-dependent mouse BaF3 cells resulted in increased Tyr15 phosphorylation of Cdc2 and enhanced cell proliferation, while cells transduced with a threonine 34 to alanine (T34A) dominant negative (DN) Survivin construct showed reduced Cdc2 Tyr15 phosphorylation and accelerated apoptosis. Furthermore, when apoptosis was induced in stably transduced BaF3 cells by IL-3 withdrawal, Survivin expression and phospho-Tyr15 levels correlated with enhanced survival and decreased survival and low phospho-Tyr15 levels correlated with Survivin disruption by T34A-DN-Survivin. Since Survivin does not contain intrinsic kinase activity we sought to determine what kinase(s) could be involved in stabilization or increase in phosphoTyr-15-Cdc2 by Survivin. Since Survivin inhibits caspase 3 activity and the Cdc2-targeting kinase Wee1 is a degradation target of active caspase 3, we performed immunoblotting assays on Survivin wt and T34A-transduced BaF3 cells and showed that cells over-expressing wt Survivin contain 2-fold higher levels of Wee1 protein as compared to those expressing vector or DN-Survivin. In addition, cells overexpressing wt Survivin contain >2-fold higher intact PARP protein levels which is another target of active caspase 3, as compared to cells transduced with MIEG vector. In order to test whether mimicking wt Survivin inhibition of caspase 3 could lead to increased Cdc2-Tyr15 phosphorylation, we evaluated the specific caspase 3 inhibitor Ac-DEVD-CHO. Inhibition of caspase 3 in BaF3 cells produced an increase in Cdc2Tyr15 phosphorylation in a dose-dependent manner, with 25 mM giving >4-fold higher intact PARP and >6-fold higher phospho-Tyr15 protein levels. Taken together, these results suggest that the antiapoptotic effect of Survivin is mediated through inactivation of Cdc2 by phosphorylation on Tyr-15 as a consequence of protection of the Wee1 kinase from degradation by caspase 3.
Hoxa9+Meis1a Efficiently Transform Hematopoietic Stem Cells but Not Committed Progenitors.
Authors: Wang Yingzi , Krivtsov Andrei , Losyev Grigoriy , Armstrong Scott A.
Published: October 2019

The homeobox transcription factors Hoxa9 and Meis1a induce the expansion of hematopoietic stem cells (HSC) and cooperate to induce acute myeloid leukemia (AML). We have recently shown that Hoxa9 and Meis1a are part of a self-renewal program found in leukemia stem cells (LSC) isolated from murine leukemias initiated by expression of MLL-AF9 in committed granulocyte macrophage progenitors (GMP). However, it remains unclear whether the Hoxa9-Meis1a complex is sufficient to mimic the leukemogenic effects of MLL-AF9. Therefore we assessed the clonogenic and leukemogenic activity of HSC and GMP transduced with Hoxa9-Meis1a and compared this to MLL-AF9 transduced HSC and GMP. We expressed either Hoxa9-Meis1a or MLL-AF9 in HSC or GMP and sorted single cells into 96 well plates. The data demonstrate that the clonogenic potential of single HSC or GMP expressing Hoxa9-Meis1a or MLL-AF9 are similar and both produce cells that can be replated in vitro for greater than 6 weeks. Remarkably, Hoxa9-Meis1a induces leukemia with high efficiency when expressed in HSC but not committed GMP, while MLL-AF9 can fully transform both HSC and GMP. Next, we identified a population of cells from Hoxa9-Meis1a leukemias that were enriched for LSC. Even though the leukemias were initiated from HSC, the LSC population possessed an immunophenotype and global gene expression program more consistent with differentiated myeloid cells. Further characterization of gene expression in the Hoxa9-Meis1a LSC found that only a subset of the MLL-AF9 self-renewal signature is activated by Hoxa9-Meis1a. These findings show that Hoxa9-Meis1a can efficiently induce LSC from HSC but not GMP. This suggests Hoxa9 and Meis1a induced leukemogenesis is dependent upon cellular context and require programs/pathways active in HSC in order to initiate leukemia. Thus MLL-AF9 must activate pathways critical for LSC development in addition to Hoxa9-Meis1a in order to fully transform committed progenitor cells. Identification of these cooperating pathways should provide insight into MLL-rearranged and other AML.
Epigenetic Regulation of CD20 Protein Expression in B-Cell Lymphoma Cells after Rituximab-Containing Chemotherapy.
Authors: Tomita Akihiro , Hiraga Junji , Kiyoi Hitoshi , Kinoshita Tomohiro , Naoe Tomoki
Published: October 2019

For the past 10 years, rituximab has been used with some forms of conventional chemotherapy to treat CD20(+) B-cell malignancies as a new molecular targeting drug. Although significant improvements in disease-free survival and cure rate have been obtained, resistance to rituximab has been recognized as a considerable problem. Here we report that CD20(−) tumor transformation is one important mechanism of resistance to rituximab. We also demonstrate a possibility that CD20 protein expression is downregulated by some epigenetic mechanisms, and interestingly, the protein expression can be restored by treatment with certain epigenetic drugs. During the 5 years beginning in 2001, 124 patients with B-cell malignancies were treated with rituximab-containing induction, consolidation, or salvage chemotherapies in our hospital. Relapse or recurrence of disease were confirmed in 36 patients (29.0%), and re-biopsy of tumor tissues was performed in 19 patients. Five cases (DLBCL, 3 cases, and DLBCL transformed from FL, 2 cases) of those 19 patients (26.3%) indicated CD20 protein-negative transformation in flow cytometry (FCM) and/or immunohistochemistry (IH) analyses. We established a CD20(−) lymphoma cell line RRBL1 from a DLBCL patient with CD20(−) transformation from CD20(+) FL after repeated treatment with rituximab, and analyzed molecular mechanisms of the CD20 protein-negative phenotype. CD20 was not expressed by IH, FCM, and immunoblotting analyses, and resistance to rituximab was observed after cell culture with rituximab treatment. Expression of wild-type CD20 mRNA was confirmed by RT-PCR, and no genetic mutation in coding sequence or promoter region was observed. Quantitative RT-PCR showed that the CD20 mRNA expression level was almost 100 times lower than that of CD20(+) B-cell lymphoma cells typically obtained from DLBCL patients. These data suggest that aberrant down regulation of CD20 mRNA may be closely related to the negative CD20 surface antigen expression. Interestingly, CD20 protein re-expression was confirmed by enhancing CD20 mRNA expression using specific epigenetic drugs including histone deacetylase inhibitor Trichostatin A (TSA). In addition, after TSA treatment, sensitivity for rituximab was significantly restored. Chromatin immunoprecipitation assay suggested that recruitment of histone deacetylase complexes to CD20 promoter may be related to the down regulation of CD20 expression. Our data may provide the basis for a new therapeutic strategy, epigenetic therapy combined with molecular targeting therapy using rituximab, for rituximab-resistant CD20(−) transformed B-cell malignancies.
Directed De Novo DNA Methylation of a Genomic Locus Leads to Heritable Transcriptional Repression.
Authors: Ford Kevin G. , Hurd Paul J. , Bannister Andrew J. , Kouzarides Tony , Smith Alexander E.
Published: October 2019

The ability to impose exogenous targeted epigenetic changes in the genome represents an attractive goal in gene therapy for the heritable repression of target genes, while potentially enabling the generation and subsequent study of the downstream effects of de novo epigenetic events, which are known to occur in disease. Here we demonstrate the ability of zinc-finger peptides to deliver DNA cytosine methylation in vivo to a genomic target promoter, when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular de novo methylation events. We show for the first time targeted gene silencing in response to directed DNA cytosine methylation via initiation of a repressive chromatin signature at a targeted genomic locus, characterised by elevation of histone H3K9Me2 and reduction of H3K4Me3 levels at that region. This transcriptional repression is maintained in the absence of sustained targeted methyltransferase action, confirming epigenetic maintenance by the cells own machinery. The inherited DNA methylation pattern is restricted to specific target sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. Therefore, we demonstrate for the first time, targeted DNA methyltransferases as potential tools for the exogenous and heritable control of gene expression at the chromosomal level, while providing the clearest and most direct confirmation to date of the functional and mechanistic consequences of de novo DNA methylation in the cell. This work represents an important step towards the longer term goal of controlling gene expression through the inheritance of a repressive DNA state, as well as providing a valuable tool for studying spatial and temporal issues associated with ‘genuine’ de novo methylation, on transcription and chromatin structure.
High-Dose Methotrexate, Ifosfamide, Etoposide and Dexamethasone (MIED) Is an Active Salvage Regimen for Children with Recurrent or Refractory Malignant Lymphoma.
Authors: Sandlund John T. , Pui Ching-Hon , Zhou Yinmei , Lowe Eric J. , Kaste Sue C. , Kun Larry E. , Krasin Matthew J. , Onciu Mihaela , Ribeiro Raul C. , Howard Scott C. , Metzger Monika L. , Relling Mary V. , Hale Gregory A. , Rencher Renee , Darnell Kim , Hudson Melissa M.
Published: October 2019

Significant advances have been made in the treatment of malignant lymphomas in children; however, approximately 20–30% will have refractory or recurrent disease. These patients are felt to have a relatively poor prognosis primarily because of the comprehensive and intensive nature of their frontline therapies; therefore, they are generally considered for a novel or more aggressive salvage regimen. The purpose of this study is to determine the activity and toxicity profile of the MIED regimen (high-dose methotrexate, ifosfamide, etoposide and dexamethasone) in children with refractory or recurrent non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). From 1991 to 2006, 62 children with refractory/recurrent NHL (n=24) and HL (n=38) were treated with 1 to 6 sequential cycles of MIED (methotrexate, 8 grams/m2 on day 1; ifosfamide, 2 grams/m2 on days 2–4; etoposide, 200 mg/m2 on days 2–4; and dexamethasone, 40 mg/m2 on days 1–4). Children with NHL also received intrathecal MHA (methotrexate, hydrocortisone, and cytarabine at age adjusted dosages) on day 1. Response evaluation was performed after 1 – 2 cycles of MIED. Children with either a PR or CR were considered for an intensification phase with hematopoietic stem cell transplantation (HSCT); patients with HL also received involved-field irradiation. Forty-six (75%) of the 61 evaluable children with refractory or recurrent lymphoma responded to MIED (CR, 23; PR, 23). Among the 24 children with NHL (large cell, 18 [anaplastic large cell, 8; diffuse large B-cell, 3; T-cell large cell, 2; large cell not otherwise specified, 5]; Burkitt, 3; lymphoblastic, 2; other, 1), responses included: CR (n=10), and PR (n=5) for a combined CR+PR rate of 63%. Among the 37 children with HL (nodular sclerosis, n = 29; mixed cellularity, n =4; lymphocyte predominant, n =1; and HL not otherwise specified, n=3) responses included: CR (n=13), and PR (n=18) for a combined CR+PR rate of 84% among 37 evaluable patients. MIED was generally well tolerated (associated with grade IV hematologic toxicity in most cases and frequently associated with mucositis and/or fever with neutropenia). Nineteen of 24 children with NHL and 31 of 37 children with HL received an intensification phase with HSCT support (autologous, 46; allogeneic, 4) at some point following MIED therapy. Nine of 24 children (38%) with NHL are alive and disease-free. Twenty-eight of 37 children (76%) with HL are currently alive (24 disease-free post HSCT). MIED is an active and generally well tolerated regimen for children with refractory or recurrent malignant lymphoma.
Diffuse Large B-Cell Lymphoma (DLBCL) with Central Nervous System (CNS) Relapse: Prognosis and Risk Factors by Retrospective Analysis from Single Center Experience.
Authors: Shimazu Yutaka , Maeda Takeshi , Notohara Kenji , Ito Takeshi , Morita Satoko , Fujiwara Yayoi , Uchiyama Tatsuki , Sato Aki , Sato Takayuki , Onishi Tatsuhito , Chisato Mizutani , Matsutama Fumio , Tsukayama Choutatsu , Ueda Yasunori
Published: October 2019

Background: The introduction of rituximab into the therapy of DLBCL has improved the prognosis dramatically. However, relapse in CNS is still the issue. We studied the prognosis and risk factors of CNS recurrence in DLBCL.

Method: Between Jan. 1996 and Apr. 2007, 441 patients were diagnosed to have DLBCL in our institute, of whom 31 patients were excluded due to CNS involvement at the time of initial diagnosis. We have analyzed 410 cases, in which 37 cases had relapsed in CNS. Before Sep. 2003, 168 patients were treated with the regimen based on CHOP, and after Sep. 2003, 242 patients were treated with the regimen based on CHOP plus rituximab. Once relapsing in CNS, the patients were treated with systemic chemotherapy plus high-dose methotrexate or radiation with intrathecal methotrexate. The risk category by the international prognostic index of these 411 cases was assessed as low: 36%, low-intermediate: 15%, high-intermediate: 23%, and high: 26%.

Results: The median age was 71 years old (range: 17–92). Median follow-up period was 507 days, and the median period free from relapsing in CNS was 331 days. The mean survival period of the cases with CNS relapse, of the cases relapsed outside the CNS, and of the non-relapsed cases was 1328 days, 2290 days, and 2817days, respectively. The overall survival rate of cases with CNS relapse was significantly lower than that of the cases relapsed outside the CNS, or than that of the non-relapsed cases (p=0.0233, p=0.0003, respectively). Multivariate Cox regression analysis identified the increased lactate dehydrogenase (p=0.014), the involvement of more than one extranodal site (p=0.006), and not using rituximab before CNS relapse (p=0.040) as an independent predictor of CNS recurrence.

Conclusion: CNS relapse has extremely poor prognosis than relapse outside the CNS in DLBCL. Rituximab may be effective in preventing CNS relapse. Since rituximab poorly penetrates into CNS, this may partly due to the reduction of all recurrence by rituximab. According to the risk assessment in CNS relapse, an effective CNS prophylaxis strategy should be determined.
Intermediate Dose Intravenous Methotrexate (MTX) Dramatically Reduces the Risk of Secondary Central Nervous System (CNS) Lymphoma.
Authors: Hochberg Ephraim P. , Hammerman Peter , Takvorian Tak , Toomey Christiana , Michaelson James , Rahemtullah Aliyah , Abramson Jeremy S.
Published: October 2019

Secondary CNS lymphoma (SCNSL) is a rare complication of diffuse large B cell non-Hodgkins lymphoma (DLBCL) which is almost universally fatal. Several groups have analyzed risk factors for the development of SCNSL and published risk estimates for both individual anatomic sites of DLBCL as well as combinations of clinical risk factors. Although the rates of SCNSL for all DLBCL patients range from 2.5% to 5% subgroups can be identified with risks as high as 33%. High risk scenarios where CNS prophylaxis is most commonly offered include either a combination of an elevated LDH and more than one extranodal site of disease, or disease involving the bone marrow, testicle, sinus, orbit, or a para-spinal location. The type of prophylaxis used varies widely from intrathecal to high-dose intravenous methotrexate as well as intrathecal use of cytarabine. Despite the advances made in the systemic therapy of DLBCL there is very limited data on the efficacy of these chemopreventive regimens in SCNSL. We undertook a retrospective review at our institution of all patients with DLBCL treated with inpatient intermediate dose methotrexate (3.5 grams per meter squared) followed by leucovorin rescue between 2002 and 2006 for CNS prophylaxis. We found 37 patients. Of 15 patients initially evaluated with diagnostic lumbar punctures (LP), no patient had a positive CSF cytology. The median age of the patients was 59 (26–79). The average IPI was 3. All patients were tested for HIV and 3 were found to be positive. The indications for CNS prophylaxis were primarily an elevated LDH and more than 1 extranodal site of disease (20) but also included patients with bone marrow (9), testicular (3), orbital (5), para-spinal (2) and sinus (6) involvement, with 6 patients having more than one site of involvement. All patients received an anthracycline containing chemotherapy regimen with curative intent. Thirty-six patients received rituximab. Patients received a median of 6 cycles of systemic chemotherapy and 3 courses of MTX given on day 15 of alternating cycles (usually 2, 4 and 6). In addition 2 patients received a single dose of 12 mg of intrathecal methotrexate at the time of their initial LP and 5 patients received intrathecal liposomal cytarabine. We calculated the expected number of cases of SCNSL using the estimates from van Besien et al. Blood ‘98, Hollender et al. Ann Onc ‘02, and Boehme et al. Ann Onc ‘06 as appropriate depending on the sites of disease for each patient. For this population the expected number of cases of SCNSL was 7.98 and the observed number of cases was 1 (p=.002 two-sided binomial probability). In this population of 37 patients with an elevated risk of SCNSL, the use of intravenous intermediate dose methotrexate substantially reduced their risk of this fatal complication.
A Multicenter Phase II Trial of Etoposide, Methylprednisolone, High-Dose Cytarabine, and Oxaliplatin (ESHAOx) for Patients with Refractory/Relapsed Aggressive Non-Hodgkin’s Lymphoma.
Authors: Sym Sun Jin , Kang Hye Jin , Nam Seung-Hyun , Kim Hoyoung , Kim Seok Jin , Eom Hyeon Seok , Kim Won Seog , Suh Cheolwon
Published: October 2019

Etoposide (E), methylprednisolone (S), high-dose cytarabine (HA), and cisplatin (P) (ESHAP) combination is commonly used salvage regimen for non-Hodgkin’s lymphoma (NHL). Oxaliplatin (Ox), a new platinum derivative, showed substantially different cytotoxic activity and adverse effects from both cisplatin and carboplatin. In addition, single-agent oxaliplatin was reportedly active in patients with NHL. We conducted to investigate the efficacy and toxicity of ESHAOx combination, substituting oxaliplatin for cisplatin in ESHAP combination, for relapsed/refractory aggressive NHL patients. Main eligibility criteria included aggressive NHL and failure to achieve a complete remission or recurrent disease after previous chemotherapy. ESHAOx consisted of E, 40 mg/m2 on days 1 to 4; S, 500 mg on days 1 to 5; HA, 2 g/m2 on day 5; and Ox, 130 mg/m2 on day 1, every 3 weeks. Eligible patients were scheduled to receive a maximum of 6 cycles, and high dose chemotherapy and hematopoietic stem cell rescue allowed. Responses were evaluated every 3 cycles. All patients gave written informed consent before study entry. Between May 2006 and January 2007, 27 patients were enrolled. Nineteen (70%) patients with relapsed, 8 patients with refractory, and 10 (37%) patients with IPI 3–5 were included in this study. A total of 102 cycles were administered for a median number of 4 (range 1–6 cycles) per patient. There were 8 (30%) complete responses and 9 (33%) partial responses, producing an overall response rate of 63% (95% CI, 45–81%). Most common grade 3/4 toxicity of the courses was myelosuppression with including neutropenia (55%) and thrombocytopenia (33%). Non-hematologic toxicity was very favorable. No significant renal and neurotoxicity was demonstrated. There was one treatment-related death due to neutropenic sepsis. The results of ESHAOx combination showed highly antitumor activity and favorable toxicity profile, suggesting it can be used as salvage regimen for relapsed/refractory aggressive NHL patients.
Denileukin Diftitox (ONTAK) Plus CHOP Chemotherapy in Patients with Peripheral T-Cell Lymphomas (PTCL), the CONCEPT Trial.
Authors: Foss Francine , Sjak-Shie Nelida , Goy Andre , Jacobsen Eric , Advani Ranjana , Smith Mitchell , Komrokji Rami , Pendergrass Kelly , Bolejack Vanessa , Watts Karen , Acosta Mark
Published: October 2019

PTCL are an aggressive group of lymphomas comprising a number of histopathologic subtypes for which CHOP chemotherapy has been the standard first-line regimen at many centers. Denileukin diftitox (Dd) is a recombinant DNA-derived cytotoxic protein composed of diphtheria toxin fragments A and B and the full length sequence of human interleukin-2 (IL-2). Dd targets T-cells expressing the intermediate and high-affinity IL-2 receptor. Because the mechanism of action of Dd is distinct from traditional cytotoxic chemotherapy and Dd has exhibited minimal myelosuppression, we evaluated the safety, tolerability, and efficacy of combining Dd with CHOP as first-line therapy for patients with PTCL according to the REAL classification. (Pts with mycosis fungoides or Sezary syndrome were not included.) Dd was administered at 18 mcg/kg/day on Days 1 and 2 followed by CHOP on Day 3, and G-CSF support starting Day 4, every 3 weeks for up to 6 cycles. Evaluation of response is performed after every 2 cycles of treatment.

Results: Forty-one pts, 18 male/23 female, have been enrolled to date, with a median age of 52. Ten pts are not evaluable for response: 5 discontinued due to an adverse event (AE) prior to assessment of response; 3 due to lack of measurable disease at baseline; and 2 are too early to evaluate. For the 31 response-evaluable patients, the overall response rate is 90%, with 71% (22/31) CR or CRu, 19% (6/31) PR, 6% (2/31) SD, and 3% (1/31) PD. Ten of 28 responders (36%) have progressed, with a median duration of response of 13 months.

PTCL subtype No. of Patients No. evaluable Responses ORR PTCL-nos 20 15 3 CR, 5 CRu, 4 PR, 2 SD, 1 PD 80% Angioimmunoblastic 10 9 3 CR, 5 CRu, 1 PR 100% Anaplastic large cell 6 4 2 CR, 2 CRu 100% Enteric T-cell 2 1 1 CRu 100% Hepatosplenic 1 0 n/a n/a Nasal/nasal type T/NK cell 1 1 1 CRu 100% Subcutaneous panniculitic T-cell 1 1 1 PR 100%

Toxicities have generally been grade 1–2, were transient, and caused few dose modifications. The most common have been fatigue (62%), nausea (44%), anemia (38%), sensory neuropathy (33%), hypoalbuminemia (33%), elevated ALT (30%), dyspnea (28%), thrombocytopenia (30%), leukopenia (28%), fever (25%), elevated AST (25%), lymphopenia (25%), and hyperglycemia (25%). Forty-three percent (17/40) of patients experienced one or more grade 3–4 hematological toxicities and 40% (16/40) of patients experienced one or more grade 3–4 non-hematological toxicities. Five patients discontinued the study due to AEs, all occurring prior to Cycle 2, for: infusion-related anaphylaxis; elevated LFTs; port-related staph sepsis; pneumonia; and a death possibly resulting from tumor lysis syndrome and rhabdomyolysis.

Conclusion: The combination of Dd plus CHOP has a generally manageable safety profile and to date has exhibited promising clinical activity in PTCL.