bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Gene Expression Patterns and Interaction Networks in BCR/ABL Positive and Negative Adult Acute Lymphoblastic Leukemias.
Authors: Juric Dejan , Lacayo Norman J. , Ramsey Meghan C. , Racevskis Janis , Wiernik Peter H. , Rowe Jacob M. , Goldstone Anthony H. , O’Dwyer Peter J. , Paietta Elisabeth , Sikic Branimir I.
Published: October 2019

BCR/ABL is associated with an unfavorable prognosis in adults with acute lymphoblastic leukemia (ALL). We used DNA microarrays to identify gene expression profiles and molecular interaction networks related to BCR/ABL status and clinical outcome in a set of 54 adult ALL specimens from the MRC UKALL XII/ECOG E2993 intergroup study (21 p185BCR/ABL- and 16 p210BCR/ABL-positive and 17 BCR/ABL negative). In order to avoid biases associated with commonly used sample amplification procedures, we have implemented an indirect two-step labeling protocol based on signal amplification by use of dendrimer technology. Using a two-class, non-parametric t-test and a false discovery rate cutoff of 5%, we identified 271 genes (including GAB1, CIITA, XBP1, CD83, SERPINB9, PTP4A3, NOV, LOX, CTNND1, BAALC and RAB21) as differentially expressed in BCR/ABL positive ALL compared with BCR/ABL negative ALL. They separate these two classes of adult ALL with an overall accuracy of 93% and are enriched for three highly relevant biological functions: Cellular Growth and Proliferation (57 genes, p = 0.004–0.044), Cell Death (49 genes, p = 0.0007–0.049), and Hematological System Development and Function (40 genes, p = 0.00004–0.049). Network analysis demonstrated complex interaction patterns of these genes, and identified FYN and IL15 as the hubs of the top-scoring network. We confirmed these findings by both qRT-PCR analysis of the initial set of samples and by cross-platform validation in an independent cohort of 128 adult ALL specimens. In addition, within the BCR/ABL positive subgroup, we identified 14 clones found to be over-expressed (TSPAN16, ADAMTSL4) or under-expressed (PILRB, STS-1, SPRY1) in p185BCR-ABL- relative to p210BCR-ABL-ALL. In a nearest-centroid classification, these clones correctly predict the BCR/ABL subtype with a cross-validated prediction accuracy of 95%. No differential gene expression was detected among Rho family GTPases and their known interaction partners. Finally, we constructed a gene expression- and interaction-based outcome predictor consisting of 27 genes (including GRB2, GAB1, GLI1, IRS1, RUNX2 and SPP1), which strongly correlated with overall survival in BCR/ABL positive adult ALL (p=0.0001), independently of other clinical parameters such as age (p=0.25) and white blood cell count at presentation (p = 0.003). These findings may be useful for developing novel therapeutic targets and prognostic markers in adult ALL.
Antibody Dependent Cellular Cytotoxicity (ADCC) Augmentation of Immunotoxin Activity in SCID-HSB-2 Mice Is Adoptively Transferable to NOD/SCID-HSB-2 Mice.
Authors: Flavell David J. , Flavell Sopsamorn U.
Published: October 2019

The anti-CD7 immunotoxin HB2-SAPORIN has significant therapeutic activity in the SCID-HSB-2 model of human T-ALL (Morland et al 1994 Br J Cancer 69, 279). In contrast the same immunotoxin shows virtually no therapeutic activity against the same tumour cell line in the NOD/SCID-HSB-2 model (Flavell et al 2000 Br J Cancer 83,1755). One difference between the two animal models is the presence of defective NK cells in NOD/SCID mice compared with the possession of fully functional NK cells by SCID mice. As we have previously shown that NK cell-mediated ADCC contributes significantly to HB2-SAPORIN activity in the SCID-HSB-2 model (Flavell et al 1998 Cancer Res 58, 5787) we hypothesise that it is defective NK cell function and a consequent reduction in ADCC in the SCID/NOD-HSB-2 model that explains the reduced therapeutic efficacy of immunotoxin in these animals. To test this hypothesis we adoptively transferred splenocytes from untreated or poly inosinic:cytidilic (poly I:C) treated SCID mice to NOD/SCID-HSB-2 mice prior to their treatment with immunotoxin. Adoptive transfer of un-stimulated splenocytes to NOD/SCID-HSB-2 mice did appear to improve the therapeutic performance of immunotoxin in these animals though this was not significant when compared to that observed in sham adoptively transferred animals. However, adoptive transfer of poly I:C activated splenocytes from SCID mice to NOD/SCID-HSB-2 mice did significantly improve the therapeutic performance of HB2-SAPORIN in the NOD/SCID-HSB-2 model. These preliminary data provide some further evidence for our hypothesis that it is deficient ADCC in the NOD/SCID-HSB-2 model that is responsible for reduced immunotoxin activity.
Effective Induction of Apoptosis by Mistletoe Plant Extracts in an Acute Lymphoblastic Leukemia Model.
Authors: Seifert Georg , Jesse Patrick , Prokop Aram , Reindl Tobias , Lobitz Stephan , Laengler Alfred , Lode Holger N. , Henze Guenter
Published: October 2019

Mistletoe (Viscum album) is one of the most used alternative cancer therapies applied as monotherapy or in combination with conventional therapies. Anti-tumor effects of mistletoe (MT) extracts were related to cytostatic and immunomodulatory effects observed in vitro. Aqueous MT extracts contain the three mistletoe lectins I, II and III as one predominant group of biologically active agents. The MT lectins inhibit protein biosynthesis by inactivating the 60S ribosomal subunit. Mistletoe lectin-I (ML-I) is one important apoptosis inducing compound. It is a heterodimer that consists of a cytotoxic A-chain (ribosome inactivating protein, RIP type 1) linked by a carbohydrate binding B-chain for cellular lectin uptake. However, although MT is widely used, there is a lack of scientific preclinical and clinical data. Here, we describe for the first time efficacy and mechanism of MT extracts against lymphoblastic leukemia in vitro and in vivo.

For this purpose, we first investigated both the cytotoxic effect and mechanism of action of two standardized aqueous MT extracts (MT obtained from fir trees (MT-A); MT obtained from pine trees (MT-P)) and isolated ML-I, in three human acute lymphoblastic leukemia (ALL) cell lines (NALM-6, sup-B-15 and REH). MT-A, MT-P and ML-I clearly inhibited cell proliferation as determined by LDH reslease assays at very low concentrations (ML-I LD50 from 0,05 ng/ml to 10 ng/ml depending on the host tree) with MT-P being the most cytotoxic extract. The mechanism of cell death was determined by DNA-fragmentation assays. These indicated dose dependent induction of apoptosis as the main mechanism of cell death.

Finally, we evaluated the efficacy of MT-A and MT-P in an in vivo SCID-model of pre-B ALL (NALM-6). For this purpose, mice (n=8/group) were injected i.v. with 1 × 106NALM6 cells and treated by intraperitoneal injections four times per week for 3 weeks (day 1–4; 7–11; 14–18) at varying doses (1, 5 and 50 mg/Kg (plant weight/body weight)). Mice (n=8) treated with PBS and cyclophosphamide (100 mg/kg, once on day 1) were used as negative and positive controls, respectively. Toxicity, peripheral blood counts, bodyweight and survival was determined over time.

Interestingly, both MT extracts in all tested concentrations significantly improved survival (up to 55,4 days) in contrast to controls (34,6 days). Furthermore, no hematologic side effects were observed from this treatment as indicated by completely stable blood counts. Also the body weight of treated animals remained stable over time indicating a complete absence of systemic toxicity in the selected dose range.

In summary, we demonstrate for the first time efficacy and mechanism of MT extracts against ALL in vitro and in vivo and hereby provide an important base line for the design of clinical trials with these compounds.
Outcome of Adult ALL Patients with Fusion Transcript E2A-PBX1: Results from the GMALL Therapy Trials 5/93 and 6/99.
Authors: Burmeister Thomas , Goekbuget Nicola , Schwartz Stefan , Hoelzer Dieter , Thiel Eckhard
Published: October 2019

The chromosomal translocation t(1;19) with fusion transcript E2A-PBX1 is the third most prevalent recurrent translocation in adult ALL (after t(9;22) and t(4;11)), accounting for about 3% of all cases. It’s prognostic impact in adults has been controversially discussed and it has been suggested that this aberration might define a high risk patient group. However, this would be in contrast to the situation in pediatric ALL, where E2A-PBX1/t(1;19) meanwhile defines a patient group with a better than average outcome. We have investigated retrospectively by RT-PCR 337 B-precursor BCR-ABL-/MLL-AF4-negative ALL patients enrolled in the GMALL 5/93 and 6/99 therapy trials for the presence of this mRNA fusion transcript. This represents the largest cohort of adult BCR-ABL-negative patients investigated for E2A-PBX1 by RT-PCR thus far. We assessed the clinical characteristics of the E2A-PBX1-positive patients and their overall survival.

Results: Eighteen patients were E2A-PBX1-positive by RT-PCR (5.8% of the investigated group, roughly 3% of the total of all ALL patients). Fourteen had a pre B (cyIg+), and 4 a common immunophenotype. Twenty-two percent of all pre B ALL patients were E2A-PBX1-positive. The median age of the positive patients at diagnosis was 27 (15–60) years and the median leukocyte count was 36.700/μl (N=17) with 7 of 17 patients presenting with an initial leukocyte count >30.000/nl (59% vs 27% in the negative group, p=0.005). Compared to the E2A-PBX1-negative group (239 common, 49 pre B, 31 pro B, median age 32 years, median leukocyte count 11.900 (N=312) the E2A-PBX1-positive patients had a lower overall survival rate (0.33 vs. 0.36). The CR rate did not differ significantly between E2A-PBX1-positive and -negative patients.

These results support the assumption that in contrast to the situation in pediatric ALL E2A-PBX1 defines a relatively unfavourable genetic subgroup in adult ALL. It is strongly correlated with high leukocyte count (>30.000/μl) at diagnosis which is a highly significant poor prognostic factor in B-precursor ALL. It remains to be established whether E2A-PBX1 can be confirmed as an independent prognostic factor within standard risk B-precursor ALL. E2A-PBX1 may be useful to separate in so called standard risk ALL a patient group which may benefit from stem cell transplantation in first CR.
Factor V Binding to Multimerin 1: Modulation by Factor V Activation and Binding Sites in the Factor V C1 and C2 Domains.
Authors: Jeimy Samira B. , Quinn-Allen Mary Ann , Fuller Nola , Segers Kenneth , Stafford Alan R. , Weitz Jeffrey I. , Camire Rodney M. , Nicolaes Gerry A.F. , Kane William H. , Hayward Catherine P.M.
Published: October 2019

Platelets and endothelial cells store the polymeric factor V(a) binding protein, multimerin 1 (MMRN1), for release upon agonist stimulation. In human megakaryocytes, factor V binding to MMRN1 follows plasma factor V endocytosis, resulting in stored complexes of MMRN1 and factor V in platelet α-granules. The C2 domain of the factor V light chain contains a MMRN1 binding site; however, the affinity and stoichiometry of factor V-MMRN1 binding have not been determined, direct comparisons of factor V and Va binding to MMRN1 have not been done, and potential homologous roles of C1 and C2 domain structures in MMRN1 binding have not been studied. To further explore the mechanism of factor V and Va binding to MMRN1, and the roles of B domain release and C1 domain residues in MMRN1 binding, we used surface plasmon resonance and solid-phase binding studies. Functional consequences of factor V-MMRN1 binding were tested in competitive binding assays with the soluble phospholipid 1,2-Dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), and calibrated automated thrombinography (CAT). Factor V bound to MMRN1 with a higher affinity than factor Va (approximately 2 nM versus 12 nM), and a stoichiometry consistent with binding to MMRN1 trimers. The higher affinity of factor V for MMRN1 was mainly due to differences in rates of formation of a more stable, secondary complex with MMRN1. Factor V activation by thrombin dissociated bound factor V from MMRN1, consistent with the reduced affinity of factor Va for MMRN1. A panel of point mutated, B domain deleted factor V constructs were used to identify MMRN1 binding residues in the C1 domain of factor V and Va. On a three dimensional model of factor Va, these residues mapped to a large, predominantly contiguous region between the C1 and C2 domains, that overlapped residues critical for factor Va phospholipid binding and procoagulant function. Consistent with the lowered affinity of factor Va for MMRN1, C6PS significantly inhibited factor Va-MMRN1, but not factor V-MMRN1 binding (p<0.05). Overlap between the MMRN1 and phospholipid binding sites was verified by CAT assays, as MMRN1 caused dose-dependent, significant reductions in plasma thrombin generation in these assays, by increasing lag time (p<0.01), and reducing peak (p<0.01) and total thrombin generation (p<0.01). Taken together, these data indicate that the functional homologies between the C domains of factor V extend to their MMRN1 binding sites. Moreover, thrombin has modulating effects on factor V-MMRN1 binding that mimic its effects on factor VIII-von Willebrand factor binding. The affinity of factor V-MMRN1 binding could be important to promote the association of MMRN1 with factor V in platelets, until factor V release and activation for prothrombinase assembly.
CD15 (Lewis x) Expression in Human Myeloid Cell Differentiation Is Regulated by Sialidase Activity.
Authors: Gadhoum Samah Z. , Sackstein Robert
Published: October 2019

The cell surface carbohydrate Lewis x (Lex, CD15) is now well established as an important marker for immunophenotyping leukocytes and for immunoregulatory functions. Lex is related to sialyl Lex (sLex) by the addition of a sialic acid in the core Lex trisaccharide. This sialylation has profound implications as it confers novel biological function(s) to the core trisaccharide. In particular, expression of sLex correlates with E-selectin ligand activity. Though the expression of Lex in myeloid differentiation is now widely recognized, no studies to date have addressed the molecular mechanism regulating its expression. To examine this issue, we employed a leukemic model of differentiation based on the capacity of anti-CD44 mAbs to induce maturation and Lex expression. We found that anti-CD44 mAb-induced expression of Lex (> 30% increase in Mean Fluorescence Intensity (MFI) by flow cytometry) was accompanied by decreased expression (> 20% decrease in MFI) of its related structure sialyl Lex (sLex). Two non-mutually exclusive processes could account for the increase in Lex expression during myeloid differentiation:neo-synthesis of the Lex epitope and/orde-sialylation of the existing sLex.

To test whether increased expression of Lex during CD44-induced differentiation resulted from augmented synthesis, Acute Myeloid Leukemia (AML) cells were treated with anti-CD44 mAb in the presence of 4F-GlcNAc (2-acetamido-1,3,6-tri-O-acetyl-4-fluoro-D-glucopyranose), an inhibitor of poly-N-acetyl-lactosamine synthesis. 4-F-GlcNAc did not significantly alter the increase of Lex induced by the anti-CD44 mAb indicating that the CD44-mediated increase of Lex does not require de-novo polylactosamine synthesis. In presence of the sialidase inhibitor DANA (2,3-dehydro-2-deoxy-N-Acetyl-neuraminic acid) the anti-CD44-induced increase of Lex and decrease of sLex were abrogated indicating that sialidase plays an important role in the modulation of Lex expression during CD44 mAb-induced granulocytic differentiation. Moreover, using the exogenous substrate 4MU-NANA to measure endogenous sialidase activity in AML cells we found that the anti-CD44 mAb Hermes-1 induced a significant increase in the sialidase activity of AML cells, strongly suggesting that CD44-induced differentiation causes the elaboration of endogenous sialidases that are responsible for the modulation of Lex level by converting sLex into Lex. Furthermore, treatment of immature myeloid cells with G-CSF induced a 2-fold increase in sialidase activity, coincident with increased morphological changes of differentiation suggesting that the induction of surface sialidase activity is associated dynamically with the conversion of immature to mature phenotypes among native myeloid cells. Our results unveil a new paradigm, demonstrating a critical role for glycosidase activity in the “biosynthesis” of a key glycan determinant expressed on human cells.
p38 MAPK Signaling Mediates Retinoic Acid-Induced Expression of a Novel Tumor Suppressor Protein Programmed Cell Death 4 (PDCD4) in Acute Promyelocytic Leukemia Cells.
Authors: Akar Ugur , Ozpolat Bulent , Colburn Nancy , Lopez-Berestein Gabriel
Published: October 2019

Programmed-cell-death-4 (PDCD4) is a novel tumor suppressor protein that suppresses tumor promoter-induced neoplastic transformation. PDCD4 specifically inhibits the helicase activity of eukaryotic translation initiation factor 4A (eIF4A) and translation initiation and cap-dependent mRNA translation in vitro and in vivo. Loss or underexpression of PDCD4 is associated with carcinogenesis and chemoresistance in solid tumors. The role and regulation of PDCD4 in the the hematopoietic system and myeloid leukemia cells are not known. We previously reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms during terminal cell differentiation (Harris et al, Blood, 104 (5) 2004). Therefore, in this study, we investigated the expression and regulation of PDCD4 during myeloid cell differentiation. All-trans-retinoic acid (ATRA) induces terminal differentiation in acute myeloid leukemia (AML) and promyelocytic (APL) cells, a well established model for myeloid cell differentiation. We found that treatment of HL60 (M2 type AML) and NB4 APL (M3 type AML) cells with ATRA (1 mM) induced PDCD4 protein and mRNA expression during granulocytic differentiation detected by western blot and RT-PCR analysis, respectively. We also demonstrated that inhibition of PDCD4 by siRNA reduced granulocytic differentiation induced by ATRA, suggesting that PDCD4 plays a role in granuliocytic differentiation. To determine mechanisms regulating PDCD4 we investigated the role of pP38 MAPK (Mitogen activated protein kinase) in reugulation of PDCD4 expression. ATRA induced PDCD4 expression correlated with activation of p38 MAPK (Mitogen Activated Protein Kinase) pathway in NB4 cells. To test the hypothesis that p38 MAPK signaling pathway mediates retinoic acid induced PDCD4 expression we treated cells with a specific p38 MAPK inhibitor, SB203580, ATRA or combination with ATRA. We observed that p38 inhibitor inhibited ATRA-induced expression of PDCD4 in NB4 cells. Basal level of PDCD4 expression was also markedly downregulated in the presence of p38 inhibitor when compared to untreated control cells, suggesting that p38 pathway is involved in ATRA-dependent and independent PDCD4 expression. Currently we are investigating whether inhibition of p38 by small interfering RNA (siRNA) will prevent expression of ATRA induced PDCD4 in APL cells. We are also trying to identify whether ATF2 transcription factor, a downstream of p38, is involved in PDCD4 expression. p38-mediated induction of PDCD4 pathway reveals a novel mechanism of PDCD4 regulation and ATRA action, providing a new insight into understanding terminal differentiation of myeloid cells. Better understanding the role of PDCD4 and posttranscriptional control of gene expression may offer targets for the differentiation therapy and chemo preventive strategies.
Crystal Structure of the C2 Domain of Lactadherin and Computational Docking of Phosphatidyl-L-Serine.
Authors: Shao Chenghua , Head James F. , Seaton Barbara A. , Gilbert Gary E.
Published: October 2019

Lactadherin is a phosphatidyl-L-serine (Ptd-L-Ser)-binding protein that decorates the membranes of milk fat globules. Lactadherin is also secreted by macrophages, engaging Ptd-L-Ser on the membranes of apoptotic cells and hastening clearance by the macrophages. Sequence homology between the lectin-like C1 and C2 domains of lactadherin and those of factor VIII and factor V correlates with the capacity of lactadherin to compete for sites on Ptd-L-Ser-containing membranes and function as a potent anticoagulant. The C2 domain of lactadherin contains a major Ptd-L-Ser binding motif. Solution studies have shown that binding of the phospho-L-serine head group exhibits stereospecificity, consistent with the existence of one or more specific binding sites on the protein. Neither the three-dimensional structure of the lactadherin C2 domain nor the identity of specific residues that interact with Ptd-L-Ser have been determined. We have solved the crystal structure of the bovine lactadherin C2 domain (Lact-C2) at 1.67 Å resolution. The β-barrel protein core, composed of eight anti-parallel strands, is homologous with the previously published structures of the factor VIII C2 domain (fVIII-C2) and factor V C2 domain (fV-C2). The loops on the lower surface of Lact-C2 display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interacting residues of fVIII-C2 and fV-C2. These hydrophobic amino acids, Trp26, Leu28, Phe31, and Phe81, form a diamond-shaped hydrophobic patch on the lower surface of Lact-C2. This hydrophobic patch is well positioned to interact extensively with the phospholipid membrane. The longest loop, predicted to participate in membrane binding, has a W-like shape that contrasts with the simpler shapes adopted by the corresponding loops of fV-C2 and fVIII-C2. Furthermore, in Lact-C2 glycine residues within putative membrane-interacting loops appear likely to provide conformational flexibility that may facilitate Lact-C2 binding to the membrane. To investigate potential Ptd-L-Ser binding sites on the protein, computational docking studies were performed, utilizing the program AutoDock. A Ptd-L-Ser analog with acetyl moieties replacing both acyl chains was utilized as the docking ligand. This approach avoided spurious identification of non-specific binding sites for acyl chains, which would naturally be immersed in a membrane bilayer. We identified two candidate Ptd-L-Ser binding sites that could engage the hydrophilic head group of Ptd-L-Ser in a stereoselective manner. The binding pocket for the better of the two solutions is not homologous with the Ptd-L-Ser binding sites previously proposed for fVIII-C2 or fV-C2. These results provide the basis for a testable hypothesis rationalizing the efficient phospholipid binding of lactadherin vs. factor VIII and factor V and the mechanism for Ptd-L-Ser stereoselectivity.
The Feasibility of Combining Daunorubicin, Clofarabine and Gemtuzumab Ozogamicin Is Feasible and Effective. A Pilot Study.
Authors: Burnett Alan K. , Kell William J. , Hills Robert K. , Russell Nigel H. , Yin John , Hunter Ann , Culligan Dominic
Published: October 2019

New treatment combinations are required for older patients with AML. Clofarabine is a novel nucleoside analogue which has been shown in several phase 2 studies in older patients to be active alone or in combination. Gemtuzumab Ozogamicin (GO) is also active in older patients and is currently undergoing evaluation in several randomised trials. As part of the proposed UK NCRI AML16 Trial for patients over 60 years we have undertaken a Pilot Trial to establish the feasibility combining standard dose Daunorubicin (D) (50mg/m2 days 1,3,5), Clofarabine (Clo) and GO (3mg/m2) as a novel combination, with the intention to compare D+Clo vs D+Ara-C both +/− GO on day 1. Five treatment cohorts were planned. Daunorubicin at a dose of 50mg/m2 is included in all cohorts. Cohorts 1–4 combine D with Clo at a dose level of 15mg/m2, 20mg/m2, 25mg/m2 and 30mg/m2 given on days 1–5 for 2 courses, to establish the feasible Clo dose in combination. Cohort 5 comprises D + feasible Clo dose given as above + GO 3mg/m2 on day 1 of course 1. Each cohort initially comprised 6 patients, and cohorts were expanded based on toxicity scoring. If toxicity criteria were satisfied the next cohort was opened. Thirty-four patients have been recruited. Of 6 patients in cohort 1 (15mg) 5 achieved CR without toxicity with one having refractory disease; of 7 patients in cohort 2 (20mg) 6 achieved CR with 1 having refractory disease; of 10 patients in cohort 3 (25mg) 4 achieved CR, 3 had refractory disease and 3 had induction deaths; and of 9 patients in cohort 4 (30mg) 5 entered CR, 2 had RD and 2 ID. Based on this, a dose of 20mg/m2 was chosen for clofarabine. So far cohort 5 has recruited 2 patients, both of whom have entered CR. This cohort will shortly be completed.

Toxicity: No toxicity of grade >2 in AST and ALT was seen in any cohort; one patient on Clo (30mg) had a transient grade 3 bilirubin elevation after courses 1 and 2. One patient (30mg) had grade 4 renal toxicity. The mean time on antibiotics was 13.1 days with no difference between the cohorts. The median time to recovery of ANC was 19 (17–24) days and platelets 19 (14–21) days.

Conclusion: The combination of Daunorubicin 50mg/m2 on days1, 3 and 5 and Clofarabine 20mg/m2 days 1–5 with Gemtuzumab Ozogamicin 3mg/m2 on day 1 is an effective and feasible combination for prospective evaluation.
A Multicenter Randomized Comparison of Maintenance Treatment with Androgens in Elderly Acute Myeloid Leukemia after ICL Regimen as Induction Therapy: Results of the Goelams-2002 Study.
Authors: Pigneux Arnaud , Himberlin Chantal , Hunault-Berger Mathilde , Witz Francis , Recher Christian , Harousseau Jean-Luc , Tournilhac Olivier , Berthou Chistian , Escoffre-Barbe Martine , Guyotat Denis , Fegueux Nathalie , Delain Martine , Lioure Bruno , Bauduer Frédéric , Jourdan Eric , Bouscary Didier , Legros Laurence , Perry Frédéric , Milpied Noel-Jean , Bene Marie-Christine , Ifrah Norbert , Sotto Jean-Jacques
Published: October 2019

In an attempt to improve the outcome of acute myeloid leukemia in older patients, we performed a multicenter randomized trial evaluating the possible benefit of androgens during post remission therapy. All patients received the ICL regimen as induction (Idarubicin, 8mg/m2 d1–5; Cytarabine, 100mg/m2 d1–7 and Lomustine, 200mg/m2 d1). All patients were randomized at the time of diagnosis to receive, after achieving CR or PR, a maintenance therapy including or not androgens. Maintenance therapy consisted of 6 courses of reinduction with idarubicin (8mg/m2 d1) and cytarabine (100mg/m2d1–5, subcutaneously) every 3 months, and, between these courses, a continuous regimen of methotrexate and 6-mercaptopurine. Patients randomized with androgens additionally received 10 to 20 mg according to body weigh of norethandrolone daily from CR/PR on, for up to 2 years. Between June 2002 and January 2005, 330 patients aged ≥ 60 years (median 70 yrs, range 60–86) were included. CR, PR, failure and death rates after induction were 69 %, 6%, 9% and 15% respectively. The median follow-up for the 330 patients was 26 months.

Among the 249 patients who achieved remission, 119 had been randomized with norethandrolone and 130 without. The 2 arms were balanced according to age, sex-ratio, performance status, FAB, WBC as well as platelets counts, peripheral blood and bone marrow blasts counts and cytogenetic groups (low risk: t(8;21), inv(16); high risk: del(5), del(7), del(11q), complex karyotype; standard-risk: normal karyotype and other aberrations). Differences in CR rates were only related to the latter parameter of cytogenetic features (P = 0.005).

For the whole cohort of patients DFS at 3-years was 23±3% (median 13 months) and OS at 3 years was 29±3% (median 15 months). For patients who achieved CR or PR and had been randomized, the addition of androgens did not influence either DFS (P = 0.6) or OS (P = 0. 9), even when considering age groups, high/low WBC, or cytogenetic features.

In conclusion, the ICL regimen followed by six reinductions and two years maintenance therapy induce high CR rates and improve OS and DFS. However the addition of androgens did not decrease the relapse rate.
Induction Therapy with Fludarabine, ara-C and G-CSF Results in Improved Event Free Survival in Core Binding Factor Leukemia.
Authors: Borthakur Gautam , Kantarjian Hagop , Wang Xuemei , Giles Francis , Cortes Jorge E. , Pierce Sherry , Estey Elihu H.
Published: October 2019

Previously (Blood2001;98:3575–3583) we reported that pts with inv (16) or t(8;21) (CBF) seemed to benefit from receiving fludarabine + ara-C (FA) rather than idarubicin + ara-C (IA), although other groups did not. Ara-C doses were 2g/m2 daily X 4–5 with FA and 1.5g/m2 daily X 2–4 by continuous infusion with IA. Because this observation was based on only 14 CBF pts given FA we proceeded, from 2000-present, to give all CBF pts FA either with (2000–2002; 22 pts) or without G-CSF (2003-present; 45 pts) on the days when FA was administered. Idarubicin(12 mg/m2 daily X 3) was given only if pts presented with WBC > 50,000, forcing start of treatment before cytogenetic results were known; once CR was achieved and CBF status was known, pts received 6 courses of post-remission therapy with FA (or FA +GCSF, FLAG) as described above. Median age was 41.5 years (range 16–83), 61 were male, 16 patients had antecedent hematologic disorder (AHD), 96 patients had ECOG performance status (PS) ≤ 1. One hundred and six patients (93%) achieved complete remission (CR). Events were defined as death, failure or relapse. Median time to event was 91 weeks and estimated EFS at 10 years was approximately 40%. Event-free survival (EFS) was compared in the 67 pts treated with FA or FLAG with EFS in 47 pts treated with IA +/− GCSF (IA/IAG). Because IA/IAG was given to CBF pts from 1991–2000 and FA or FLAG from 2000-present, we compared covariate-adjusted EFS in 436 pts with cytogenetics other than CBF according to whether they received IA before or after 2000 to estimate “period-effect”.EFS was superior in the earlier period, and this difference, seeming to inherently favor IA/IAG rather than FA or FLAG, was accounted for in the comparisons of these treatments in the CBF group. Preliminary analyses suggested that EFS in CBF pts was similar between IA and IAG (these groups were considered together for subsequent analyses) and longer with FLAG than with FA or IA/IAG (p= 0.06). After adjustments for covariates (age, WBC,platelets, marrow blasts etc) we compared EFS with FA, FLAG ,and IA/IAG, using both standard (Cox regression) and Bayesian approaches. The Bayesian model indicated that the posterior probability that FLAG was superior to IA/IAG was 0.98, and that, among pts given FA, the posterior probability that FLAG was superior to FA was 0.91. The Cox model indicated that the risk of an event with FLAG relative to IA/IAG was 0.84 (p = 0.07). A possible explanation for the seeming superiority of FLAG is the ability of G to increase accumulation of fludarabine-triphosphate in blasts during therapy (Clin Cancer Res1995; 1:169–178).

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Generation of Acute Myeloid Leukemia (AML)-Specific Autologous CD4 and CD8 T Cell Lines by Limiting Dilution AML Dendritic Cell Culture.
Authors: Zhong Rui-kun , Lane Thomas A. , Ball Edward D.
Published: October 2019

Naturally occurring cytotoxic T cells directed against various leukemia associated antigens (LAA) expressed by acute myeloid leukemia (AML) cells have been described. However, these LAA-specific T cells are rare and obviously unable to initiate effective anti-leukemia responses. The challenge is how to investigate, select, activate and expand the rare LAA-specific T cells from the vast population of blood cells in patients with AML for immunotherapy. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we have developed a novel method of generating multiple autologous AML reactive T cell lines by limiting dilution AMLDC (LD-AMLDC) culture. The principle of LD-AMLDC is based on the assumption that autologous AML-reactive T cells or precursors are randomly distributed in the AML PBMC suspension, and that each one has an equal opportunity to respond to AML cells in the 96-well plates under optimized culture condition. By culturing AML PBMC (>90% blasts) in culture medium supplemented with GM-CSF/IL4/IL2/IL7/IL12 to induce AML DC differentiation and activate in situ autologous T cells, highly reactive anti-AML T cell lines (both CD4+ and CD8+ lines) were selected and expanded from LD-AMLDC culture using the appropriate numbers of AML PBMC in each culture well by the criterion of release of IFN-gamma in response to autologous AML blasts. By maximum likelihood solution, the estimated average frequency of AML reactive T cells or precursors is 6±3/1,000,000 AML PBMC (n=8). Strong intracellular IFN-gamma release of T cell lines obtained in LD-AMLDC was demonstrated by flow cytometry analysis after stimulation by autologous AML cells but not autologous B-lymphoblastoid cell line (LCL) (Figure). Effective specific lysis (up to 70% at E:T=20:1) of autologous AML cells but not autologous LCL or allogeneic AML cells by these T cell lines was observed. Two PR1 specific T cell lines were obtained by screening 39 AML reactive HLA-A2+ CD8+ T cell lines generated from 5 LD-AMLDC cultures, suggesting that other unidentified CD4 or CD8 lines with strong autologous AML responses may be reactive to known or unknown LAAs. These results encourage continued efforts to induce, activate and select T cells lines with high autologous AML reactivity using LD-AMLDC culture and to expand multi-LAA reactive T cell lines acquired from limiting dilution AML-DC culture for AML immunotherapy.

Figure Figure
The Expression of T Cell Receptor Signaling Genes Is Associated with Poor Response to IFN-α and/or PUVA in Mycosis Fungoides.
Authors: Wozniak Magdalena , Ortiz Pablo , Tracey Lorraine , Peralto Jose L. , Alvarez Monica , Piris Miguel A. , Villuendas Raquel
Published: October 2019

Mycosis fungoides (MF) is a low-grade cutaneous T-cell lymphoma, in which malignant T cell clones (mostly CD4+) arise in the skin from the early disease stages. IFN-α is widely used in the treatment of MF and when used in combination with PUVA has been reported as an effective treatment, with overall response rates of 30%–80%, and complete response rates of 14%–25%.

However, up to date there is no information available on prognostic factors that could help to predict response to IFN-α /PUVA in MF. The purpose of the study was to find the molecular signature associated with IFN-α /PUVA resistance, or lack of remission after IFN-α /PUVA treatment. The gene expression profile of the pre-treatment samples from 30 MF patients enrolled in a random clinical trial with IFN-α and/or PUVA has been analyzed by use of cDNA microarrays. Following the treatment outcome, the patients have been divided into good responders (23 patients that have achieved complete remission in the time of 24 weeks or less) and bad responders (7 patients that have not reached completed remission or have shown progression of the disease during treatment). The genes associated with good vs. bad response have been identified. Four genes associated with cell cycle regulation and tumour microenviroment have been identified to predict good response by the significance analysis of microarrays (SAM) correlating expression data with survival time. Moreover, 38 genes involved in T cell receptor signaling pathway, NF-kB activation and Jak-Stat signaling pathway have been found to be associated with unfavorable response to treatment by use of SAM analysis. This was validated using other bioinformatics tools based on t-statistics and Cox-model, applying False Discovery Rate for multiple testing. Furthermore, using a web-based tool (Signs) that uses a combination of gene filtering, clustering and survival model building, a 2-gene model has been obtained. This model could distinguish two groups of MF patients with probability of remission at 24 weeks of 15%, and 60% (log-rank test, p:0.007). This confirms that TCR-signaling plays a key role in cutaneous T-cell lymphoma cell survival; and could potentially be used for stratifying MF patients treated with IFN-α /PUVA into different risk-groups, if confirmed in additional studies.
The Oncogenic Fusionprotein NPM-ALK Cooperates with Bcl2 To Induce T-Cell Lymphomas in Mice.
Authors: Miething Cornelius , Grundler Rebekka , Mugler Claudia , Bauer Anette , Häcker Georg , Peschel Christian , Duyster Justus
Published: October 2019

The NPM-ALK fusion oncogene arising from the t(2;5) translocation has been shown to transform lymphoid cells in culture and induce tumors in mice. In a previous report, we have demonstrated that expression of NPM-ALK in a murine retroviral infection/transplantation model leads to histiocytic and plasmacytoid tumors in mice. In the earlier model, we did not find any signs of a T-cell malignancy in the transplanted animals, although control animals transplanted with an empty vector expressing EGFP demonstrated successful targeting of the T-cell compartment. To further delineate the effects of NPM-ALK expression in T-cells in cell culture, we infected a human T-cell line (Jurkat) with NPM-ALK. Interestingly, NPM-ALK expressing cells demonstrated an increased rate of apoptosis, whereas control infected cells showed normal growth without significant cell death. Bcl2 levels were reduced in the NPM-ALK expressing cells and exogenous Bcl2 expression could partially rescue NPM-ALK induced cell death. We therefore investigated the effect of increased Bcl2 expression in vivo by infection of either C57B6 wild type or vav-Bcl2 transgenic mice derived BM cells with NPM-ALK and subsequent transplantation into lethally irradiated recipient mice. Whereas the control mice mostly developed the previously described histiocytic malignancy without significant T-cell involvement, all mice receiving NPM-ALK and Bcl2 coexpressing cells displayed a thymus derived T-cell lymphoma with infiltration of the thymus, lymphnodes, spleen and to a lesser extent also the bone marrow, where the histiocytic phenoype predominated. The malignant T-cells expressed Thy1.2, CD4 and NPM-ALK, but were mostly TdT negative. Our results implicate that Bcl2 cooperates with NPM-ALK in the induction of T-cell lymphomas, suggesting that NPM-ALK may require additional hits to suppress apoptosis and induce full transformation in T-cells.
Genetics of t(11;14)(q13;q32)-Negative MCL.
Authors: Wlodarska Iwona , Vanhentenrijk Vera , Pospisilova Helena , Van Roosbroeck Katrien , Stul Michel , Minnei Francesca , Vandenberghe Peter , Verhoef Gregor , De Wolf-Peeters Chris
Published: October 2019

Mantle cell lymphoma (MCL) is hallmarked by IGH-mediated t(11;14)(q13;q32) resulting in deregulation of CCND1. This gene encodes cyclin D1 that together with cyclin D2 and -D3, plays a key role in cell cycle progression. Given that cyclin D1 is not expressed by normal B lymphocytes, its aberrant expression in lymphoma has a diagnostic value. Recently, it has been shown that rare cyclin D1-negative MCL cases express either cyclin D2 or cyclin D3. The molecular mechanisms underlying these events are unknown. We report here the genetic characteristics of 12 MCL cases without cytogenetic and FISH evidence of CCND1 rearrangement. These cases were collected over the last 15 years; they displayed the typical morphology and gene expression profile (determined by comparative expressed sequence hybridization) of MCL. Of interest, 5 of these lymphomas revealed cerebral or meningeal lesions at diagnosis or during a course of disease. Immunohistochemistry and RT-PCR studies showed a lack of expression of D-type cyclins in 2 cases. One of these cases was documented by t(2;14)(p24;q32) targeting NMYC, as shown by FISH and qRT-PCR. In this case upregulation of NMYC possibly led to inactivation of Rb and an aberrant progression of G1/S transition. The second case was characterized by a complex karyotype including loss of 17p. In the remaining 10 cases expression of cyclin D1, -D2 and -D3 in respectively 3, 2 and 5 cases was found. Cytogenetics and FISH identified t(2;12)(p11;p13) resulting in the IGK-CCND2 rearrangement in 1 of the 2 cases expressing cyclin D2 and der(14)t(6;14)(p21;q32) and der(20)t(14;20)(q32;p13) in one of the cases expressing cyclin D3. The breakpoint at 6p21 however was mapped distal to CCND3. FISH studies of p16, p27, p53 and Rb performed on all cases except one, identified loss of one copy of 1 to 4 of the analyzed genes in 7 cases. These data support the existence of t(11;14)-negative MCL that frequently express one of the three D-type cyclins. In one of the cases a new IG translocation affecting NMYC was found. Other genetic aberrations possibly involved in pathogenesis of t(11;14)-negative MCL included chromosomal translocations affecting CCND2 and unidentified genes at 6p21 and 20p13, and loss of tumor suppressor genes involved in cell cycle regulation.
Elevated Apurinic/Apyrimidinic Endonuclease Activity Significantly Contributes to DNA Instability in Multiple Myeloma.
Authors: Shammas Masood A. , Bertheau Robert C. , Koley Hemanta , Neri Paola , Tassone Pierfrancesco , Batchu Ramesh B. , Munshi Nikhil C.
Published: October 2019

Apurinic/apyrimidic (AP) sites are the commonest DNA damage lesions and are highly mutagenic and cytotoxic requiring immediate repair. AP endonucleases (APE) 1 and 2 are the key proteins involved in the repair of AP sites and therefore play an important role in maintaining genomic integrity. However, if APE activity is dysregulated, it may lead to increase in DNA breaks leading to genomic rearrangements. In this study we demonstrate that endonuclease activity as measured by a novel plasmid based assay is elevated over 5 fold in myeloma cell lines and primary myeloma cells relative to normal plasma cells. APE1 and 2 were identified as the major endonuclease in MM by gene expression profiling and protein studies as well as using methoxamine (MX), which specifically inhibits APE activity by reacting with the aldehyde group of an abasic site. To further investigate the role of elevated APE activity in MM, we cultured MM cells in the presence of MX and demonstrate a significant inhibition of DNA recombination activity, as well as acquisition of new mutations as assessed by change in genome-wide loss of heterozygosity (LOH) using the 100K single nucleotide polymorphism arrays. Conversely, in cell line with lower APE activity we demonstrate a significant increase in genomic rearrangement with acquisition of significant number of new LOH loci. These data suggest that dysregulated AP endonuclease activity plays an important role in ongoing accrual of mutational changes, which may be associated with progression of myeloma and/or development of drug resistance. Moreover APE1 and APE2 provide a novel target to prevent disease progression and drug resistant phenotype associated with genomic instability.
Pro-Apoptotic Effect of Lenalidomide (L) in Patients with Chronic Lymphocytic Leukemia (CLL) Is Possibly Mediated through Interruption of the Phosphatidylinositol Pathway.
Authors: Chanan-Khan Asher Alban , Ersing Noreen , Krammer Deborah , Bernacki Ralph , Coignet Lionel , Goodrich David , Bangia Naveen , Padmanabhan Swami , Porter Carl W.
Published: October 2019

Introduction: In a phase II clinical trial we reported the antileukemic effect of L in CLL pts. In this clinical trial 24/34 (70.5%) pts with detectable tumor cells (ALC > 5,000) in peripheral blood demonstrated a decrease in ALC within 8 days of treatment. Although, the exact molecular mechanism for its antitumor activity remains undetermined, L has been reported to down regulates production of various cytokines including VEGF, IL-6 and TNF-alpha. In order to investigate the underlying mechanism(s) responsible for the antileukemic effects of L in CLL, we examined down stream targets of VEGF and IL-6 signaling pathways in CLL cells obtained from pts pre and post 8 day treatment with L.

Method: Pts with accessible tumor cells in blood (ALC > 5000) who received L were identified. Tumor cells were obtained prior to (baseline) and 8 days after treatment with L. Whole cell lysate was prepared and expression of down stream targets of VEGF, AKT and Erk determined through immunobloting assay.

Results: In cells obtained from pts who had received 8 days of treatment, we observed that L decreased the expression of pAKT and pErk1/2 without changes in corresponding total protein. Conversely L did not have any effect on protein expression of the Bcl-2 or Bcl-xl, though Mcl-1 was decreased compared to pretreatment control.

Conclusion: This is the first time that the antileukemic effect of L in CLL may at least be attributed to down regulation of VEGF and IL-6 mediated prosurvival pathways. Our observation that L does not interrupt the Bcl-2 pathway are clinically intriguing and propose the possibility of combining L with Bcl-2 inhibiting agent(s) to augment the antitumor response in CLL.
Jak2: A Potential Therapeutic Target for Chronic Myelogenous Leukemia (CML).
Authors: Samanta Ajoy K. , Lin Hui , Sun Tong , Kantarjian Hagop , Arlinghaus Ralph B.
Published: October 2019

In most CML patients Bcr-Abl, a constitutively active tyrosine kinase derived from the Philadelphia chromosome, is highly expressed and is the causative factor in most CML patients. Imatinib mesylate, an inhibitor of the Bcr-Abl kinase, is a very effective drug for treatment of CML. However in some CML patients, drug resistance develops and the patients relapse. Thus, alternative drug targets need to be identified. We have shown that Bcr-Abl activates its downstream target, the Jak2 tyrosine kinase, leading to the enhancement of c-Myc expression (Xie et al. Oncogene21: 7137, 2002; Samanta et al. Cancer Res.66: 6468, 2006). Our recent studies showed that Bcr-Abl activated the transcriptional factor NF-kB through Jak2, which in turn activated c-Myc transcription. Jak2 also activated Akt, which increased c-Myc protein levels by inhibiting GSK3. Addition of AG490, an inhibitor of the Jak2 kinase, prevented enhanced expression of c-Myc and caused induction of apoptosis in BCR-ABL+ leukemia cells. Immunoprecipitation experiments showed that Bcr-Abl is associated with a cluster of signaling proteins including Jak2, Gab2, Akt and GSK3b. Treatment of CML cell lines and mouse BCR-ABL+ 32D cells (myeloid lineage) with the either Jak2 siRNA or the Jak2 kinase inhibitor AG490 caused inhibition of pTyr Gab2 formation, pSer Akt formation and the activation of NFkB. Of interest, treatment of BCR-ABL+ 32 D cells with IL-3 reversed the apoptotic effects of imatinib by activation of Jak2 even though Bcr-Abl was inhibited. Importantly, mouse BaF3 hematopoietic cells expressing the T315I and E255K imatinib-resistant mutants of BCR-ABL underwent apoptosis upon exposure to either the Jak2 inhibitor AG490 or siRNA for Jak2, yet were resistant to imatinib. Cells from a number of CML patients (including six chronic phase, one accelerated phase, and two blast crisis patients who failed imatinib treatment) were induced to enter apoptosis upon treatment with AG490, whereas normal samples were not affected by AG490. Further analysis of imatinib resistant Bcr-Abl cell lines showed that transfection of the cells with Jak2 specific siRNA or by treating the cells with AG490 reduced levels of pLyn, pAkt, c-Myc and pGSK3 level compared to untreated cells. Transfection of Lyn specific siRNA into K562 and 32Dp210 cells resulted in down-regulation of pGab2, pAkt, pGsk3 and c-Myc, but did not alter pJak2 levels; this result indicates that pLyn is downstream of Jak2 but upstream of Gab2, pAkt, pGSK3 in BCR-ABL+ leukemia cells. We hypothesize that Jak2 activation of Lyn tyrosine kinase in BCR-ABL+ leukemia cells leads to tyrosine phosphorylation of the YxxM motif of Gab2, which activates the PI-3 kinase-Akt pathway. In conclusion, since inactivation of Jak2 inhibits many of the critical oncogenic targets of Bcr-Abl (resulting in apoptosis induction), we propose that Jak2 is a potential therapeutic target for CML, in both imatinib sensitive and imatinib resistant patients.