bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of CD20 Expression in Rituximab-Sensitive (RSCL) and Rituximab Resistant Cell Lines (RRCL) Using IL-4 and Bryostatin-1.
Authors: Tsai Ping-Chiao , Hernandez-Ilizaliturri Francisco J. , Olejniczak Scott H. , Naveen Bangia , Czuczman Myron S.
Published: April 2020

While the use of rituximab in combination with chemotherapy resulted in an improved survival among various subtypes of B-cell lymphomas, a significant number of patients fail to respond or relapse as a consequence of intrinsic or acquired resistance. A change in CD20 antigen density expression is a potential mechanism to explain rituximab resistance. Moreover, several groups of investigators are focused in understanding the mechanisms that regulate CD20 expression and to develop therapeutic strategies to up-regulate CD20 expression (i.e. IL-4, GM-CSF or Bryostatin-1). Up-regulation of CD20 in DB and Ramos cells by Bryostatin-1 was found to be PKC and Erk dependent. In an attempt to characterize the mechanisms responsible for rituximab resistance we developed several RRCL derived from rituximab-sensitive RL and Raji cells. We have demonstrated a significant down-regulation of CD20mRNA and CD20 surface antigen in RRCL when compared to RSCL. In our present work we evaluated the mechanisms involved in the mRNA down-regulation of CD20 and the potential of IL-4 and Bryostatin-1 in modulating CD20 antigen expression among a panel of RRCL. To this end, RSCL and RRCL were treated with either 5ng/ml of IL-4 or 3 different doses of Bryostatin-1(1, 3 or 5ng/ml). Nuclear and cytosolic extract were also obtained from RSCL RL and RRCL RL-4RH after 24, 48 and 74 hrs exposure to IL-4 (5ng/ml) or control. Differences in the expression of key regulatory transcription factors for B-cell lymphocyte development (PU.1, Oct-2, Pax5, E2A and EBF) were studied by Western Blotting. Previously, we found a significant down-regulation of CD20 antigen in the RRCL. An up-regulation of cytosolic and surface CD20 was detected in RRCL exposed to either IL-4 or Bryostatin by western blotting and flow cytometric analysis. Besides, a higher expression of Pax5, PU.1, EBF was found in nuclear fractions of RRCL when compared to RSCL. In vitro exposure of RRCL to IL-4 decreases the expression of PU.1, Oct-2, Pax5 and EBF in nuclear extracts from RSCL or RRCL when compared with controls treated cells. Our data suggest that rituximab resistance is associated with the up-regulation of transcription factors PU.1, Oct-2, Pax5 and EBF and concomitant suppression of CD20 antigen. Future study of how these agents induce CD20 expression in RRCL will provide a potential means to reversing resistance towards rituximab treatment.
Myc Induces Acute Myeloid Leukemia by Conferring Self-Renewal Activity to Committed Myeloid Progenitor Cells That Resist Apoptosis Via Endogenous Mcl-1.
Authors: Luo Hui , Cain Jennifer A. , Molitoris AnnaLynn , Opferman Joseph , Tomasson Michael H.
Published: April 2020

Ectopic expression of Myc in most primary cell types induces apoptosis, and cancer development typically requires additional, anti-apoptotic mutations. We reported previously that ectopic expression of Myc in unfractionated murine bone marrow cells induced rapid onset acute myeloid leukemia (AML) without detectable anti-apoptotic mutations. We hypothesized that AML developed in our model because a subset of normal primary bone marrow cells were inherently resistant to Myc-induced apoptosis. Consistent with this model, seven days of Myc activation in the bone marrow of mice caused the reduction of B-lineage cells while at the same time inducing the expansion of myeloid lineage cells. We sought to determine the mechanism by which myeloid progenitor cells evaded Myc-induced apoptosis, and found that Myc-induced AML cells exhibited a distinct profile of pro- and anti-apoptotic proteins, including high levels of the anti-apoptotic Bcl-2 family member Mcl-1. To prioritize apoptosis genes, we examined AML patient microarray data and found MCL1 to be uniformly expressed at high levels in human AML (94/94, 100%). We used Mcl1 heterozygous mice (Mcl1F/null) as bone marrow donors for transduction-transplantation experiments and found that, compared with Mcl1 wild-type (median survival=60 days), haploinsufficiency for Mcl1 completely protected mice from Myc-induced AML (median survival not reached). Mice transplanted with Mcl1F/null cells co-expressing Myc and Bcl2 succumbed rapidly to disease (median survival 25 days). In wild-type mice, defined hematopoietic stem and myeloid progenitor cell populations were not significantly increased by Myc activation. However, Myc transduction conferred serial replating ability to sorted hematopoietic stem and progenitor cells including lineage-committed (Lin+Kit+) progenitors cells. These data demonstrate a critical role for Mcl1 in our AML model and suggest that dysregulation of MYC in MCL1-expressing progenitor cells may mediate AML pathogenesis in humans.
HOX Genes - Candidate Tumor Suppressor Genes in Adult and Childhood Leukemia.
Authors: Strathdee Gordon R. , Holyoake Tessa L. , Sim Alyson , Parker Anton , Oscier David G. , Melo Junia V. , Meyer Stefan , Eden Tim , Dickinson Anne M. , Mountford Joanne , Jorgensen Heather G. , Soutar Richard , Brown Robert
Published: April 2020

The role of the HOX gene family in leukemia development has been extensively studied. However, these studies have focused almost exclusively on the potential oncogenic role of HOX gene family members. In contrast to the oncogenic function often attributed to HOX genes, our studies have identified several HOX gene family members as candidate tumor suppressor genes and shown that inactivation of HOX genes, particularly HOXA4, is associated with poor prognosis. We have used multiple quantitative methylation assays to search for epigenetic inactivation of HOX genes in adult and childhood leukemia. In both adult myeloid and lymphoid leukemia two members of the HOXA cluster (HOXA4 and A5) were found to be frequently inactivated by promoter hypermethylation (26–64% of cases were hypermethylated). In contrast, a further 12 HOXA, B and C cluster genes were found to be essentially devoid of hypermethylation (except HOXA6 in CLL, where 34% of samples exhibited hypermethylation). HOXA4 and HOXA5 were also frequently inactivated in childhood ALL and AML (39–79% of samples). However, in contrast to the adult leukemias, all but one of the additional HOX genes analyzed were also found to be targets for hypermethylation in both ALL and AML (4–26% of samples), suggesting that HOX genes are differentially regulated in childhood versus adult leukemia. Hypermethylation of HOX genes (HOXA4, HOXA5 and HOXA6) was associated with loss of expression of the corresponding gene. Expression analysis also suggests that interaction between different HOX genes may be crucial. In normal karyotype AML samples, those expressing of high levels of HOXA9, but not those with low HOXA9 expression, were associated with invariable HOXA4 hypermethylation (p=0.01). Interestingly HOXA4 hypermethylation also correlates with poor prognosis in all types of leukemia tested. Hypermethylation of HOXA4 correlates with progression to blast crisis (p=0.007) and poor response to imatinib in CML (p=0.04), with cytogenetic status in AML (33%, 72% and 100% in good, intermediate and poor prognostic groups respectively, p=0.0004) and with IgVh mutational status (p=0.003) and poor survival in CLL (median survival 159 versus 199 months in hypermethylated and non hypermethylated patients, respectively). Furthermore transfection of a HOXA4 expressing construct into a CML blast crisis cell line results in re-expression of markers of myeloid differentiation, suggesting that loss of HOXA4 is functionally relevant in leukemic cells. These results indicate that aberrant epigenetic regulation of HOXA4, and indeed other frequently inactivated HOX genes such as HOXA5 and HOXA6, may play a key role in the development of multiple types of leukemia. Thus co-ordinated up and down regulation of expression of HOX gene family members may be crucial in the leukemogenic process.
CDC4 and NOTCH1 Mutations in Childhood T-Cell Acute Lymphoblastic Leukemia and T Cell Non-Hodgkin’s Lymphoma; a Japan Association of Childhood Leukemia Study Group.
Authors: Park Myoung-ja , Taki Tomohiko , Suzuki Nobuhiro , Oda Megumi , Yagi Keiko , Kobayashi Ryoji , Hara Juniti , Horize Keizo , Hayashi Yasuhide
Published: April 2020

Risk-adapted therapy has resulted in a improvement of outcome in childhood acute lymphoblastic leukemia (ALL), however, T-cell ALL (T-ALL) have still relatively poor outcome compared to B-precursor ALL. Although activating mutations of the NOTCH1 gene have been reported in more than half of T-ALL, the prognostic significance of this finding has yet to be determined. CDC4 gene was isolated as a negative regulator of NOTCH1 and mutations were detected in T-ALL cell line and other cancers. We performed mutation analysis of the NOTCH1 and CDC4 gene in 14 T-ALL cell lines, 52 T-ALL and 17 T cell non-Hodgkin’s lymphoma (T-NHL) fresh samples. All children with T-ALL and T-NHL were treated on Japan Association of Childhood Leukemia Study (JACLS) ALL-97 and NHL-98 protocol after obtaining informed consent. Mutation detection was performed via PCR based denaturing HPLC followed by direct sequence.Mutations of the NOTCH1 were identified in 10 (71.4%) of 14 T-ALL cell lines, 16 (30.8%) of 52 T-ALL and 6 (35.3%) of 17 T-NHL fresh samples. Twelve mutations in heterodimerization domain (HD) and 12 mutations in PEST domain (PD) were found in 69 fresh samples. The incidence of the NOTCH1 mutation is less frequent than that of previous reports. We found CDC4 mutations in 12 (35.3%) of 52 T-ALL and 2 (11.8%) of 17 T-NHL fresh samples. One insertion mutation in exon 3 and 11 missense mutations were detected in CDC4 gene. Both NOTCH1 and CDC4 mutations were found in 7 patients. Interestingly, the NOTCH1 mutations were only observed in T-ALL and T-NHL patients without relapse, suggesting to be associated with favorable prognosis. However, CDC4 gene was found not to be associated with prognosis.
Use of Chromosome Engineering To Model a Leukemia-Associated 7q22 Deletion in the Mouse.
Authors: Wong Jasmine C. , Zhang Yan , Tran Marie T. , Lieuw Kenneth H. , Wolff Linda , Killeen Nigel , Shannon Kevin
Published: April 2020

Identifying tumor suppressor genes from intervals that are deleted in human hematologic malignancies such as 5q, 7q, 9q, and 20q has proven extremely challenging, and several laboratories have implicated haploinsufficiency as a likely mechanism. Chromosome engineering, which involves performing sequential rounds of gene targeting to insert loxP sites at flanking loci in mouse embryonic stem (ES) cells and using Cre recombination to delete the intervening sequences, was recently used to successfully interrogate the 1p36 interval in human solid tumors (Cell128(3):459–75, 2007). Monosomy 7 and deletion 7q [del(7q)] are among the most common cytogenetic alterations found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Cytogenetic analysis of patients who developed myeloid disorders with del(7q) uncovered a 2.5 Mb commonly deleted segment (CDS) within 7q22 (Blood88(6):1930–5, 1996), suggesting that this region plays an important role in leukemogenesis. To investigate the in vivo consequences of somatic loss of this interval, we generated a 5A3flox mouse model that harbor loxP sites flanking a ∼2 Mb interval on mouse chromosome 5A3 that is syntenic to the human 7q22 CDS. We intercrossed these mice with the interferon inducible Mx1-Cre transgenic strain, and injected these mice with polyinosinic-polycytidylic acid (pIpC) to delete the region in the hematopoietic compartment. The desired recombination is relatively inefficient; however, hematopoietic cells with loss of this region persist in the stem/progenitor compartment for over 1 year and are transplantable. We neither observed a block in differentiation nor clonal outgrowth of mutant hematopoietic cells, suggesting that additional mutations are necessary to initiate leukemia. We initiated AML in these mice by introducing additional genetic lesions using retroviral insertional mutagenesis, and we are characterizing these leukemias to study the effects of the 5A3 deletion on leukemogenesis and to clone cooperating genes. Chromosome engineering is a robust strategy for modeling leukemia-associated deletions in vivo, and for interrogating how loss of a specific interval alters hematopoietic growth. We are using the 5A3 strain to analyze candidate myeloid tumor suppressor genes from chromosome 7q and to uncover genes and pathways that cooperate in leukemogenesis.
Transcriptional Regulation of Hepcidin by Iron.
Authors: Lee Pauline , Jaroslav Truksa , Peng Hongfan , Beutler Ernest
Published: April 2020

Transcriptional regulation by iron in mammalian systems is poorly understood. Hepcidin, a 25 amino acid peptide that plays a central role in iron homeostasis, is transcriptionally regulated by iron. A region of the murine hepcidin promoter 1.6 to 1.8 kb upstream from the start of translation was recently identified to be important in transcriptional regulation by iron (Truksa J, et al. The distal location of the iron responsive region of the hepcidin promoter. Blood DOI 10.1182/blood-2007-05-091108, 2007). In order to identify transcription factors that might be important in regulation by iron, transcription factor microarray analyses (Panomics TranSignal Protein/DNA Array) were performed with nuclear extracts from livers of mice made iron deficient or iron loaded for 4 weeks. The analyses revealed 43 transcription factors that were upregulated in iron loaded liver nuclear extracts and 39 transcription factors that were upregulated in iron deficient nuclear extracts. In the region of the promoter we had found essential for transcriptional regulation by iron, −1.6 to −1.8 kb, consensus motifs were identified by Genomatix MatInspector for 10 transcription factors that corresponded to transcription factors upregulated in high iron nuclear extracts by array analyses. Similarly, the consensus sequences for 5 transcription factors corresponded to transcription factors identified in iron deficient nuclear extracts. Electrophoretic mobility shift assays were performed with probes across this region of the murine hepcidin promoter. Several probes exhibited differential binding between deficient and high iron nuclear extracts. These include the probe encompassing the CCAAT box and MEL1 motif, a probe containing a HLH motif, and a probe containing a bZIP and COUP motif. The probe containing the CCAAT motif was supershifted with antibodies against CBF, but was not supershifted with antibodies against SMAD4, CEBPα, and COUP. The probe containing a bZIP and COUP motif can be supershifted with antibodies against COUP-Tf and HNF4α, but not with antibodies against SMAD4, CEBPα, and COUP. Our data suggest that CBFA, COUP, and HNF4α are involved in transcriptional regulation of hepcidin by iron.
Liver Iron Measurement by SQUID Biosusceptometry Compared to Liver Biopsy: A More Accurate Definition of Optimal Iron Range.
Authors: Pakbaz Zahra , Fischer Roland , Vichinsky Elliott , Fagaly Robert L. , Paulson Douglas , Pratt Kevin , Butz 5 John , Foote Drucilla , Quirolo Keith , Harmatz Paul
Published: April 2020

The “Optimal Liver Iron” range in iron overloaded patients with thalassemia has been established by Olivieri and Brittenham (Blood, 1997) as 1–2.2 mg/g wet weight liver using direct measurement or calculated as 3.2 to 7 mg/g dry liver weight based on a 3.33 correction factor for body water. This report describes the relationship between liver iron concentration (LIC) measured by SQUID biosusceptometry (BLS, wet weight) compared to liver biopsy (dry weight), in patients with thalassemia (THAL) or sickle cell disease (SCD). A total of 38 chronically transfused patients (THAL n=19, SCD n=19) were prospectively assessed for LIC measured by BLS and liver biopsy within 2 month of each other. Most BLS results were submitted to data repository before receiving iron concentration from liver biopsy. LIC was measured by a low temperature SQUID biosusceptometer system (Ferritometer®) under the standardized Hamburg-Torino-Oakland protocol. Iron in fresh tissue and paraffin embedded liver biopsy samples was measured at Mayo clinics by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Subjects ranged in age from 5 to 40 years (median: 18 y). Median LIC (n=38) measured by SQUID was 1777 (365–4883) [mg/g wet weight], median LIC by biopsy (fresh tissue) was 10861(1854–32864) [mg/g dry weight]. After excluding the subjects with BMI>30 kg/m2 (n=5), the Spearman rank correlation between wet weight LIC assessed by BLS and dry weight LIC from fresh tissue sample measured by ICP-MS was highly significant (RS=0.90, p<0.0001). No significant difference between fresh tissue and paraffin embedded LIC values was observed. The agreement between BLS and fresh tissue biopsy was tested by Bland-Altman plots. After normalization, no significant proportional or zero bias was visible. The 95% limits of agreement between SQUID-BLS and biopsy LIC values were found between −56% (or –6733 μg/g) and 48% (or 7027 μg/g). This kind of agreement is similar to the results achieved with MRI-R2 by St. Pierre et al (Blood, 2005)(–56% and 50%), however, BLS is a totally independent method relying only on the specific magnetic susceptibility of the hemosiderin-ferritin iron complex. The conversion factor between the two methods (LIC by fresh tissue biopsy vs. LIC by BLS) obtained from a weighted linear regression with zero intercept (Marquardt algorithm) was 6.1 ± 0.3 (R2 = 0.86–0.88, cn2 = 2.3–2.6). Using this conversion factor, the recommended “optimal range” for liver iron in patients with thalassemia major should be considered as 6–13.2 mg/g dry weight liver rather than the generally accepted 3.2–7 mg/g dry weight liver.
Identification of a Protein S-Interactive Site on the Factor VIII A2 Domain.
Authors: Takeyama Masahiro , Nogami Keiji , Soeda Tetsuhiro , Yoshioka Akira , Shima Midori
Published: April 2020

Protein S functions as a cofactor of activated protein C that inactivates factor VIII(a) and factor V(a). We recently have reported a new regulatory mechanism that protein S interacted with both the A2 and A3 domains in factor VIII, and consequently this cofactor directly impaired the factor Xase complex by competing the interaction of factor IXa to factor VIIIa (Blood2006; 108, 487a). Since factor IXa blocked the binding of A2 subunit to protein S, we attempted several approaches to localize the protein S-interactive site(s) on the factor VIII A2 domain. An anti-A2 monoclonal antibody (mAb413) with the 484–509 epitope, recognizing a factor IXa-interactive site on the A2, inhibited the A2 binding to immobilized protein S up to approximately 90% in a dose-dependent manner in a surface plasmon resonance-based assay. Furthermore, ELISA-based assay showed that a synthetic peptide corresponding to residues 484–509 directly bound to protein S dose-dependently. Covalent cross-linking was observed between the 484–509 peptide and protein S following reaction with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) using SDS-PAGE. The cross-linked product formed with EDC was consistent with 1:1 stoichiometry of reactants, suggesting specificity in the 484–509 peptide and protein S interaction. This cross-linking formation was blocked by the addition of the 484–497 peptide, whilst not by the 498–509 peptide, supporting the presence of protein S-interactive site within residues 484–497. Furthermore, N-terminal sequence analysis of the 484–509 peptide-protein S product showed that three sequential basic residues (S488, R489 and R490) could not be detected, supporting that three residues participate in cross-link formation. To confirm the significance of these residues in A2 domain for protein S-binding, the mutant forms of the A2 domain, converted to alanine, were expressed in baculovirus system and purified. Compared with wild type A2 (Kd: ∼9 nM), each binding affinity of S488A, R489A, or R490A A2 mutant for protein S was decreased by 4∼5-fold (32, 40 and 40 nM, respectively). These results indicate that the 484–509 region in the factor VIII A2 domain, and in particular a cluster of basic amino acids at residues 488–490, contributes to a unique protein S-interactive site.
Immunogenicity of Factor VIII: Is Clotting the “Danger” Signal?.
Authors: Skupsky Jonathan , Scott David W.
Published: April 2020

It is well established that mice lacking factor VIII (E16 or fVIII−/−) respond to intravenous (i.v.) or intraperitoneal (i.p.) injections of therapeutic doses of human fVIII in PBS. This leads to both T-cell priming for cytokine production and inhibitor formation against fVIII with specificity similar to that of hemophilia A patients (Pratt et al., 2004). The lack of tolerance to murine fVIII may be important in the ability of fVIII−/− mice to respond to human fVIII. Nonetheless, the ability to respond effectively in the absence of extrinsic adjuvant begs the question of what is the “danger signal” required for immune responsiveness to fVIII. Even more surprising is the fact that mice with normal hemostasis respond to therapeutic doses of human fVIII given intravenously (i.v.), a normally tolerogenic route (Lin, Soukhareva, and Scott, ASH 2004). We proposed to investigate whether fVIII has any intrinsic adjuvanticity. Preliminary experiments failed to support the notion that fVIII was a polyclonal B cell activator or could stimulate up regulation of MHC class II or co-stimulatory molecules in vitro or in vivo. We hypothesized that the immunogenicity of fVIII is due to its ability to initiate clotting and that this cascade leads to local co-stimulatory events. To test this, fVIII−/− (C57Bl/6 background) or normal Balb/c mice were injected with either native (N) fVIII or heat inactivated (HI) fVIII (56o, 30′). This heat inactivation method was verified to destroy biologic activity of fVIII in chromogenic assays. After five injections, the antibody response elicited by NfVIII was three- to fourfold higher than that elicited by HIfVIII. ELISA assays with monoclonal antibodies against different fVIII domains (a gift of Dr. Pete Lollar) indicated a partial loss of binding to conformational B cell epitopes in HIfVIII. T cell proliferation to both preparations of fVIII demonstrated conservation of T cell epitopes. Importantly, the T cell response to fVIII was dramatically reduced in the group immunized with HIfVIII. These results suggest that the ability of fVIII to initiate a localized clotting event plays a critical role in the immunogenicity of fVIII.

(Supported by NIH grants R01 HL061883 and T32 HL07698).
Dasatinib Inhibits the Growth of Neoplastic Human Eosinophils (EOL-1) through Targeting of FIP1L1-PDGFRα.
Authors: Baumgartner Christian , Gleixner Karoline V. , Gruze Alexander , Samorapoompichit Puchit , Esterbauer Harald , Pickl Winfried F. , Sillaber Christian , Valent Peter
Published: April 2020

Chronic eosinophilic leukemia (CEL) is a myeloproliferative disorder characterized by molecular and/or cytogenetic evidence of monoclonality of eosinophils, sustained marked eosinophilia, and consecutive organ damage. In a majority of patients with CEL with or without associated mastocytosis, the transforming mutation FIP1L1-PDGFRα and the related CHIC2 deletion is found. The respective oncoprotein, FIP1L1-PDGFRα, is considered to play a major role in malignant cell growth in CEL. The tyrosine kinase (TK) inhibitor imatinib (STI571) has been described to counteract the TK activity of FIP1L1-PDGFRα in most patients, and has been introduced as a novel effective therapy in CEL. However, not all patients with CEL show a response to imatinib. Therefore, several attempts have been made to identify other TK inhibitors that counteract growth of neoplastic eosinophils in CEL. We provide evidence that dasatinib, a multi-targeted kinase inhibitor, blocks the growth and survival of EOL-1, an eosinophil leukemia cell line carrying FIP1L1-PDGFRα. The effects of dasatinib on proliferation of EOL-1 cells were dose-dependent, with an IC50 of 0.5–1 nM, that was found to be in the same range compared to IC50 values produced by imatinib. Dasatinib was also found to induce apoptosis in EOL-1 cells in a dose-dependent manner (IC-50: 1–10 nM). The apoptosis-inducing effects of dasatinib on EOL-1 cells were demonstrable by light microscopy, flow cytometry, and by a Tunel assay. To further examine the mechanism of growth inhibition induced by dasatinib in neoplastic eosinophils, Western blot experiments were performed using antibodies directed against phosphorylated or total PDGFRα. In these experiments, we were able to show that dasatinib at 1 μM completely blocks the phosphorylation of FIP1L1-PDGFRα in EOL-1 cells. In summary, our data show that dasatinib inhibits the growth of leukemic eosinophils through targeting of the TK activity of the disease-related oncoprotein FIP1L1-PDGFRα. Based on this observation, dasatinib may be considered as a new interesting treatment option for patients with CEL. As dasatinib is also known to block various KIT mutants as well as wild type KIT, such therapy may also be of interest for patients who have systemic mastocytosis (SM) with an associated CEL (SM-CEL).
Clinical Characteristics of T-Cell Lymphoma Associated Hemophagocytic Syndrome: Comparison of T-Cell Lymphoma with and without Hemophagocytic Syndrome.
Authors: Tong Hongyan , Ren Yanling , Xiao Feng , Mai Wenyuan , Meng Haitao , Qian Wenbin , Jin Jie
Published: April 2020

T-cell lymphoma associated hemophagocytic syndrome (T-LAHS) has been regularly reported in Asia countries and is considered with extremely poor prognosis. The rate of definite diagnosis during early stage is low and the therapeutic outcome has been disappointed. We therefore compared T-cell lymphoma patients with and without hemophagocytic syndrome (HPS) in order to have a better understanding of the clinical characteristics of T-LAHS. One hundred and thirteen patients (66 men and 47 women, age from 12 to 80 years with the median age of 42) with aggressive T-cell lymphoma admitted to our department between January 2000 and December 2005 were included in this study, while 28 of them were with T-LAHS. The patients were divided into LAHS group and no-LAHS group. The clinical data including clinical manifestations and laboratory findings were compared between the two groups by using Chi-square test. The method of Kaplan and Meier was used to analyze overall survival (OS). The results showed that LAHS occured in about 1/4 of all the patients with T-cell lymphoma, which were all aggressive type. The elevated rates of lactate dehydrogenase (LDH) and ferritin were much higher in LAHS group than in no-LAHS group. β2-microglobulin and ovarian cancer antigen (CA125) were also elevated in both groups, but there was no significant difference. The rate of hypo-fibrinogen and liver dysfunction were higher in LAHS group than that in no-LAHS group. The rate of bone marrow infiltration in LAHS group is remarkably higher than that in no-LAHS group (57% vs 32%, p<0.05). The median survival was 40 days (16 days - 22 months) in the LAHS group, and the median survival of 11 patients accepted chemotherapy more than 2 courses was 6 months. By contrast, the 2-year survival for no-LAHS group was 43%. There was significant difference between the two groups. Three patients undergoing plasmapheresis as initial therapy had survived for 3–6 months. These results indicate that high suspicion is required for early diagnosis of T-LAHS. In patients with fever, hepatosplenomegaly and cytopenia, simultaneously with serum markers such as LDH, ferritin, TG, CA125, and β2-microglobulin constantly increasing, T-LAHS should be considered. For patients without extranodal invasion or enlargement of lymph nodes, repeating biopsy of multiple sites of bone marrow may help improving the diagnosis rate. As for treatment, other more intensive regimens were not superior to CHOP regimen. While the overall outcome of treatment is still unsatisfied, plasmapheresis as initial therapy is worth considering.
Thalidomide Combinations Improve Response Rates; Results from the MRC IX Study.
Authors: Morgan Gareth J. , Davies Faith E. , Owen Roger G. , Rawstron Andrew C. , Bell Sue , Cocks Kim , Gregory Walter , Jackson Graham , Drayson Mark T. , Jenner Matthew W. , Child James A.
Published: April 2020

Improving response rates represents an important way to improve PFS and OS in myeloma. In the MRC Myeloma VII study, now with a median follow up of 8 years, we showed the importance of achieving CR. The addition of thalidomide represents a way of increasing response rates. In the MRC Myeloma IX study, which has recruited 1800 patients, in two randomisations embracing younger and older patients, we have compared CTD with MP and CTD + HDM + ASCT with CVAD + HDM + ASCT. In addition to giving insight as to the impact of thalidomide on ORR and CR in both settings, we can assess the effects of HDM + ASCT following CTD induction. We have used a number of techniques to assess response in this study including protein electrophoresis, immunofixation and serum free light chain estimation combined with bone marrow aspiration and trephine assessment. This approach has been supplemented by sensitive multiparameter flow cytometry MRD analysis able to quantitate low levels of residual myeloma plasma cells. In a preliminary analysis, a thalidomide containing combination is associated with improved response rates. Post induction rates are CTD: ORR 95.7%, CR 20.3%; CVAD: ORR 83.4%, CR 11.7% and these differences are maintained at 100 days post HDM, CTD + HDM: ORR 98.7%, CR 58.2%; CVAD + HDM: ORR 95.7, CR 41%, showing that the ORR rate is higher and that the percentage of CR is doubled before HDM and 20% more following the HDM. The depth of CR is also greater in the cases receiving thalidomide. We are updating these results as well as the results from the MP vs CTD comparison and will present these data. There are some discrepancies between each of the disease monitoring approaches and optimally all should be used. At d+100 it is possible to have true CR measured by flow MRD assessment but still have detectable paraprotein. The opposite also occurs: apparent CR by paraprotein estimation with MRD positivity by flow. This has important implications for the definition of remission as well as for the assessment of new treatments and their use. Our findings suggest that thalidomide combinations significantly improve ORR, taking more patients to response levels which render them eligible for HDM + ASCT. CR rates are increased and there is a qualitative difference in the depth of CR obtained. The additive value of HDM + ASCT, even when thalidomide is used initially, is suggested by the further increase in the number of responses above that achieved by CTD alone. We await the follow up of patients entered into this large study to assess whether these increased and enhanced responses translate into improved PFS and OS.
Dose Definition for Intravenous Cyclophosphamide in Combination with Bortezomib/Dexamethasone for Remission Induction in Patients with Newly Diagnosed Multiple Myeloma.
Authors: Kropff Martin , Liebisch Peter , Wand Hannes , Weisel Katja , Gann Claudia-Nanette , Knop Stefan , Einsele Herrmann
Published: April 2020

This study was designed to determine the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), safety, and efficacy of intravenous cyclophosphamide (CY) in combination with the proteasome inhibitor bortezomib and high-dose dexamethasone (DEX) for remission induction in younger patients with newly diagnosed multiple myeloma (MM). Patients ≤ 60 years were enrolled to receive three 3-week treatment cycles with bortezomib 1.3 mg/m2/dose on days 1, 4, 8, and 11 in combination with DEX 40 mg/day orally on the day of bortezomib injection and the day thereafter. In addition, patients received CY intravenously on day 1 at either 600, 900, 1200, or 1500 mg/m2 - the optimal dose had to be defined. Recommended concomitant medication included bisphosphonates, antacids, prophylactic antiviral treatment, prophylaxis against pneumocystis pneumonia, and oral non-absorbable antifungal medication. In addition, antiemetics, cytokines, and intravenous immunoglobulins were allowed. DLT was defined using the National Cancer Institute common terminology criteria for adverse events, version 3.0 as grade 4 hematologic toxicity, ≥ grade 3 non-hematologic toxicity (except emesis, vomiting, and alopecia), ≥ grade 3 peripheral neuropathy, and ≥ grade 2 peripheral neuropathy with pain. MTD was defined as the highest dose studied for which the incidence of DLT was ≤ 33%. Thirty patients completed 78 treatment cycles. Twenty seven patients were evaluable for CY dose definition. Start dose level for CY was 1200 mg/m2. Since no patient experienced a DLT the dose level was increased to 1500 mg/m2. On this dose level a DLT occurred in 2/3 patients (67%). Therefore the dose level was decreased again to 1200 mg/m2 with 4/12 (33%) of patients experiencing DLT. On the next lower dose level a DLT was observed in 8% (1/12). Thus, the MTD for CY in combination with bortezomib and DEX based on cycle 1 was defined as 900mg/m2. Dose-limiting toxicities included leukopenia (n=5), neutropenia (n=1), and pneumonia (n=1). No patient died. Other adverse events not reaching dose-limiting intensity included thrombocytopenia, gastrointestinal irritations, fatigue, neuropathy and one patient with herpes zoster. Median time to best response was 63 days. The overall response rate (EBMT criteria) to up to three cycles of this combination was 87%, with 3 complete responses, 20 partial responses and 3 minor responses. No patient experienced progressive disease. Bortezomib combined with DEX and CY is a highly effective treatment for newly diagnosed MM. Recommended phase II/III dose of CY in this combination is 900 mg/m2. This schedule is currently being evaluated as pretransplant induction in newly diagnosed MM in a prospective trial of the Deutsche Studiengruppe Multiples Myelom (DSMM).
Chromosomal Abnormalities in the Early Stem Cell Population of CML Patients in Complete Cytogenetic Remission.
Authors: Bumm Thomas G.P. , Newell Amy Hanlon , Oost Jay , VanDyke Jonathan , Olson Susan B. , Druker Brian J. , Deininger Michael W.N.
Published: April 2020

Clonal cytogenetic abnormalities, most commonly involving chromosomes 7 and 8, are detectable by conventional karyotyping in Ph-negative metaphases of some chronic myeloid leukemia (CML) patients with a major cytogenetic response (MCyR) to imatinib. It is unknown whether these abnormalities involve the primitive progenitor cell compartment, and whether their frequency in this compartment may exceed the frequency detected by karyotyping. To answer these questions we analyzed lineage-negative CD34+/CD38− and CD34+/CD38+ cells from CML patients in complete cytogenetic response (CCyR), using by fluorescent in situ hybridization (FISH) for chromosome 7 and 8 abnormalities and BCR-ABL.

Methods: Mononuclear cells (MNC) were selected from the bone marrow of patients with CCR by Ficoll-Hypaque density gradient centrifugation and enriched for lineage-negative cells using an immunomagnetic column. Lineage-negative cells were further sorted into CD34+/38− and CD34+/38+ cells by multicolor FACS. Interphase FISH analysis was performed using 7 LSI D7S522 Spectrum Orange / CEP7 Spectrum Green (chromosome 7), CEP8 Spectrum Aqua (chromosome 8) and LSI BCR/ABL +9q34 TriColor Dual Fusion Probe. Thus far, 5 CML patients with CCR (3 with a normal karyotype and 2 with trisomy 8) and 1 normal control have been analyzed.

Results: Of the three CML patients in CCR with normal karyotype, one had 9% deletion 7q (internal cut off of 0.5%) and the second 1.2% trisomy 8 (just over the internal cut off of 1%) cells in the CD-34+/38− population, while the CD-34+/38+ cell population did not show abnormalities. The third CCR patient had no abnormalities in the CD-34+/38− and CD-34+/38+ cell populations. Two CML patients in CCR with 45% trisomy 8 abnormal cells by conventional cytogenetics had 44% and 60% trisomy 8 positive cells in the CD34+/38+ population (two few CD34+/CD38− cells were available for analysis). No BCR-ABL signal was detected in any cell. In the healthy control, the CD34+/38+ cells were normal, but 4.1% of CD34+/38− showed a deletion of 7q.

Conclusion: Clonal chromosomal of chromosomes 7 and 8 in Ph-negative primitive hematopoietic progenitor cells may be more common than suggested by conventional karyotyping. A larger cohort of CML patients in CCR and healthy individuals is under study to determine if this phenomenon is indeed related to CML or occurs also in normals. Results will have implications for the interpretation of karyotypes in patients with hematologic malignancies.
A Phase II Trial of Myeloma Induction Therapy with Cyclophosphamide, Bortezomib, and Dexamethasone (Cybor-D): Improved Response over Historical Lenalidomide-Dexamethasone Controls.
Authors: Reeder Craig B. , Reece Donna E. , Fonseca Rafael , Bergsagel P. Leif , Hentz Joseph , Pirooz Nicholas A. , Boesiger Jacy E. , Pisa Jesus G. , Kukreti Vishal , Chen Christine , Trudel Suzanne , Mikhael Joseph R. , Lacy Martha , Rajkumar S. Vincent , Stewart A. Keith
Published: April 2020

Background: We previously reported an 85% response rate with 54% CR/nCR using a combination of cyclophosphamide (Cy), prednisone, and bortezomib (BZ) in relapsed myeloma (MM). We now report a Phase II clinical trial using a combination of Cy, BZ, and dexamethasone (Dex) [Cybor-D] in newly-diagnosed MM. We then compare the activity of this combination with historical controls treated with our current induction standard of Lenalidomide-Dexamethasone (L-Dex).

Methods: 30 patients have been enrolled on Cybor-D. Treatment consists of Cy 300mg/m2 p.o. days 1, 8, 15, and 22; BZ 1.3 mg/m2 days 1, 4, 8, and 11, and Dex 40mg p.o. days 1–4, 9–12, 17–20. Prophylactic use of acyclovir, a quinolone and antifungal prophylaxis was highly recommended for all patients on study. Growth factors were allowed. As a relevant contemporaneous control for speed and depth of response, we used 33 patients treated on a recent Mayo Clinic trial of L-Dex (Rajkumar. Blood: 2005 106;4050–3). Response was defined according to IMWG criteria although in the current trial, bone marrows for confirmation of CR are not yet available on all patients.

Results: At time of current analysis, 17 patients on Cybor-D were evaluable through at least one cycle for response and toxicity. Baseline characteristics included mean age of 57, 29% female, 41/24% ISS stage II/III. There were statistically more early stage patients on L-Dex; otherwise, baseline patient characteristics were similar. The mean % decline in monoclonal (M) protein from baseline following cycles 1–4 of Cybor-D were 71%, 90%, 93%, 96%. This compares favorably with L-Dex where equivalent % decline in M proteins were 66%, 77%, 80%, and 79%. Thus, in preliminary results, Cybor-D produces a more rapid initial decline and mean % reduction in M protein than L-Dex. Overall response rate (≥ PR) after one cycle of Cybor-D was 77% with 36% obtaining ≥VGPR (L-Dex 73% and 18% respectively). The % of patients obtaining ≥VGPR after each cycle was 36%, 64%, 77%, and 100% for Cybor-D as compared to 18%, 36%, 42%, and 33% for L-Dex. The overall ≥VGPR at the end of four cycles of Cybor-D is currently100% (≥ 66%nCR using older nomeclature). Major toxicities ≥ grade 3 for Cybor-D included hematologic (37%), hyperglycemia (15%) and neuropathy (11%).

Conclusions: In summary, for newly-diagnosed MM patients, four cycles of Cybor-D produces more frequent, rapid and deep responses when contrasted with historical L-Dex controls. Further confirmation of response rates and effect on TTP will be required.
Deletion of Murine Kininogen Gene 1 (mKNG1) Causes Loss of Plasma Kininogen and Delays Thrombosis.
Authors: Merkulov Sergei , Komar Anton A. , Schmaier Alvin H. , Barnes Ellen , Zhou Yihua , Lu Xincheng , Lou Guangbin , McCrae Keith R.
Published: April 2020

High molecular weight kininogen (HK) plays an important role in the assembly of the plasma kallikrein-kinin system. While the human genome contains a single copy of the kininogen gene, three copies exist in the rat (one encoding K-kininogen and two encoding T-kininogen). Here, we confirm that the mouse genome contains two homologous kininogen genes, mKng1 and mKng2. These genes are located on chromosome 16 in a head to head orientation with ∼ 30 kB intervening sequence, and are expressed in a tissue-specific manner. To determine the roles of these genes in murine development and physiology, we disrupted mKng1, which is expressed primarily in the liver. mKng1−/ − mice were viable, and immunoblotting using anti-bradykinin antibodies indicated a marked reduction in plasma HK and low molecular weight kininogen (LK), as well as ΔmHK-D5, a novel kininogen isoform that lacks kininogen domain 5. Clotting studies were also consistent with marked HK deficiency. Moreover, despite normal tail vein bleeding times, mKng1−/− mice displayed a significantly prolonged time to carotid artery occlusion following Rose Bengal administration and laser induced arterial injury. These results suggest that a single gene, mKng1, is responsible for production of plasma kininogen, and that plasma high molecular weight kininogen may contribute to induced arterial thrombosis in mice.
SHIP-1 Differentially Regulates Dense Granule Secretion in Platelets through Its Association with Protein Kinase C δ.
Authors: Chari Ramya , Kim Soochong , Murugappan Swaminathan , Daniel James L. , Kunapuli Satya P.
Published: April 2020

Collagen-induced glycoprotein (GP) VI-mediated and thrombin-induced protease activated receptors (PAR)-mediated activation are important signaling pathways regulating dense granule secretion in platelets. Protein kinase C (PKC) isoforms play a crucial role in platelet secretion and we have previously shown that PKCδ plays a ying-yang role in dense granule release by different agonists (Murugappan et al, J. Biol. Chem. 2004). PKCδ isoform positively regulates PAR-mediated platelet dense granule release, whereas it negatively regulates GPVI-mediated dense granule release. In this study, we investigated the mechanism of such differential regulation by PKCδ downstream of PAR and GPVI pathways. We hypothesize that the differential association of PKCδ with phosphatases downstream of GPVI and PAR receptors differentially regulate dense granule secretion. More specifically, we explored the functional relevance of the interaction of PKCδ with Src homology 2-domain containing Inositol Phosphatases (SHIP), 5′-inositol phosphatases in platelets. In our studies, SHIP-1 was tyrosine phosphorylated by both PARs and GPVI receptors and its phosphorylation followed different activation kinetics. Whereas PAR-mediated SHIP-1 phosphorylation (Y1020) was delayed and occurred as late as 120 seconds, the GPVI-mediated SHIP-1 phosphorylation was rapid, starting as early as 15 seconds and peaked at 60 seconds. Co-immunoprecipitation experiments revealed that SHIP-1, and not SHIP-2, associated with PKCδ upon stimulation of platelets with GPVI agonist, convulxin. However, such association did not occur with the PAR agonists. GPVI-mediated SHIP-1 phosphorylation failed to occur in platelets from mice lacking Lyn kinase suggesting a role for Lyn in regulating SHIP-1 phosphorylation. In murine platelets lacking either Lyn or SHIP-1, dense granule secretion was potentiated by convulxin and not by thrombin. We attribute the phosphorylation and association of SHIP-1 with PKCδ to be critical for the regulation of agonist-induced dense granule secretion in platelets. Based on the above results, we conclude that the preferential association of SHIP-1 with PKCδ upon stimulation of GPVI receptor results in the negative regulation of collagen-induced dense granule release in platelets.
Loss of Sema4D Signaling in Platelets Impairs the Formation and Stability of Arterial Thrombi In Vivo and Reduces Myocardial Infarct Size.
Authors: Zhu Li , Stalker Timothy J. , Wang Tao , Jiang Hong , Kumanogoh Atushi , Kikutani Hitoshi , Gruber Peter , Brass Lawrence F.
Published: April 2020

Contact-dependent signaling between platelets helps to promote thrombus growth and stability. One mechanism for contact-dependent signaling involves the binding of cell surface ligands to corresponding receptors on the surface of adjacent cells. In our efforts to identify novel participants in this process, we have recently reported that platelets express on their surface the semaphorin family member, sema4D, and its two known receptors, CD72 and plexin-B1 (Zhu, et al, PNAS, 2007). We have also shown that although their initial tail bleeding time is normal, platelets from sema4D(−/−) mice have a defect in collagen-induced signaling and platelet aggregation in vitro. In the present studies, we used matched sema4D(−/−) and wild type (WT) mice to examine the consequences of impaired sema4D signaling in models of platelet function in vivo. In the first model, irradiated Rose Bengal dye was used to produce an arteriolar injury in an exteriorized cremaster muscle. Platelets were identified with a fluorescent CD41 antibody and detected in real time using digital microscopy. The results showed that thrombus formation occurred in all of the mice that were tested, but while stable occlusion was observed in approximately half of the control mice, none of the sema4D(−/−) mice developed stable occlusions during the period of observation (p<0.02). Similarly, when a laser was used to produce a focal injury in cremaster muscle arterioles, both the initial rate of platelet accumulation and the peak extent of accumulation were approximately 50% lower in the sema4D(−/−) mice than in the matched controls. To test the contribution of sema4D to platelet responses in a larger artery, the right common carotid was injured by transient exposure to FeCl3 and changes in flow were measured using a Doppler probe. The results showed that the time to occlusion was 35% greater in the sema4D(−/−) mice than in controls (p<0.02). Furthermore, stable occlusion occurred in only 9 of 16 (56%) sema4D(−/−) mice Vs. 7 of 9 (78%) WT mice. Finally, myocardial infarct size was measured in an ischemia/reperfusion injury model 48 hrs after transient ligation of the left anterior descending coronary artery. Although infarction occurred in all cases, infarct volume was 56% smaller in the sema4D(−/−) mice than the matched controls (p<0.01). In summary, these results show that there is a substantial impairment of platelet function in vivo in mice that lack sema4D. This impairment was observed in both arterioles and arteries using several different methods to evoke platelet activation. When combined with our earlier observations, the results show that signaling by sema4D and its receptors provides a novel mechanism to promote thrombus growth and stability.