bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of microRNA Patterns in Hodgkin’s Lymphoma (HL).
Authors: Navarro Alfons , Gaya Anna , Pons Aina , Abrisqueta Pau , Gel Bernat , Martinez Antonio , Lopez-Guillermo Armando , Martinez Carmen , Granell Miquel , Artells Rosa , Jansa Sonia , Urbano-Ispizua Alvaro , Montserrat Emili , Monzo Mariano
Published: April 2020

Mature microRNAs (miRNA) are recently discovered small RNA molecules of 21–25 nucleotides in length. They act as negative regulators of expression of important genes like those participating in cellular proliferation or apoptosis. There is evidence that miRNA play an important role in carcinogenesis. The objective of this study was to compare the miRNA expression patterns in normal lymph nodes and in lymph nodes from patients with HL. Moreover, we investigated the miRNA pattern of HL depending on Epstein-Barr Virus (EBV) expression. We assessed 156 mature miRNAs by Stem-loop RT-PCR and Real time PCR in ABI PRISM 7500 in 9 normal lymph nodes and 39 patients diagnosed with HL nodular sclerosis subtype (15 EBV+ and 24 EBV-)at a single institution. Patients median age was 27 years (range, 15–52), and clinical stage was I (n=1); II (n=22); III (n=8) and IV (n=8); 41% of the patients reported B symptoms. RNA was obtained in all cases from formalin fixed paraffin embedded tissues. miRNA expression data was normalized to let-7a miRNA and to global median. Relative quantification of miRNA expression was calculated with the 2−ΔΔCt method. The data were presented as log10 of relative quantity of target miRNA. Normal human lymph node tissue was used as calibrator for all samples. Data were analyzed by means of Significant Analysis of Microarrays (SAM), Student’s t-test (with random variance model) and Class prediction methods using BRB Array Tools version 3.4.0 and TIGR Multiexperiment Viewer version 3.1. Of the 156 miRNAs analyzed, 35 were differentially expressed between normal lymph nodes and HL (12 miRNAs were upregulated and 23 downregulated). The most differentially overexpressed miRNAs was miR-216, which inhibits apoptosis pathway. Other differentially expressed miRNAs were miR-140, 204, 19a, 20, 191 and 142-3p, which have been associated to the genesis of hematological and non-hematological malignances. With respect to EBV+ vs. EBV− cases, we found that miR-96 and miR-335 were underexpressed in the EBV+ cases as compared to EBV− (p=0.001). These miRNAs have RNF34 and BIRC2 genes as targets, which have an antiapoptotic function. We also found that miR-138 was more frequently overexpressed in clinical stages I–II versus clinical stages III–IV (p=0.004), and that miR-328 was more frequently overexpressed in stages III–IV (p=0.004). In conclusion, miRNAs might have a role in the pathogenesis of HL. The miRNA pattern is different between the EBV+ and EBV− cases, and the differentially expressed miRNAs seems to be related to the apoptotic pathway.
Alemtuzumab in Combination with CHOP and ESHAP as First-Line Treatment in Peripheral T-Cell Lymphoma.
Authors: Intragumtornchai Tanin , Bunworasate Udomsak , Nakorn Thanyaphong Na , Rojnuckarin Ponlapat
Published: April 2020

Patients diagnosed with peripheral T-cell lymphomas (PTCL) generally had a poorer prognosis compared to B-cell non-Hodgkin’s lymphomas. With conventional treatment, the 5-year overall and failure-free survivals (OS and FFS) were 36% and 23%, respectively (Vose et al, Blood2005;106:abstract 811). Between February 2005 and January 2006, 13 consecutive patients newly diagnosed with PTCL (5, extranodal nasal NK/T-cell lymphoma, 4 subcutaneous panniculitis-like, 3 PTCL, unspecified and 1 enteropathy type) were enrolled. The median age was 44 years (range, 21–56) and male:female was 1.6:1. Fifty-four percent had stage III/IV, 31%, PS 2–3, 69%, B-symptoms, 15%, bulky disease, 46%, > 1 extranodal site, 38%, elevated serum LDH and 39%, aaIPI 2–3. Twenty-three percent had thrombocytopenia. Patients were treated with alemtuzumab 30 mg. sc. D1-3 of cycle 1–5 plus CHOP (day 1 of cycle 1, 3, 5) and ESHAP (day 1 of cycle 2, 4, 6) at 28-day intervals. Valacyclovir 500 mg tid and trimethoprim/sulfamethoxazole were given for prophylaxis of CMV and Pneumocystis carinii infection, respectively. Of the evaluable 10 patients, complete remission was obtained in 8 patients, 1 had partial remission and 1 had CNS progression while on treatment. Infection was a major adverse complication: 54% had CMV reactivation (1 had CMV disease), 54%, febrile neutropenia and 15%, tuberculosis. With a median follow-up time of 8 months, the 2-year OS and FFS were 75% (95%CI, 41–92) and 48% (95%CI, 14–76), respectively. From the standpoint of this result, alemtuzumab in combination with CHOP and ESHAP is an effective front-line therapy for patients newly diagnosed with PTCL.
Fc Engineered Anti-CD19 Monoclonal Antibodies with Enhanced In Vitro Efficacy Against Multiple Lymphoma Cell Lines.
Authors: Zhukovsky Eugene A. , Chu Seung , Bernett Matthew , Hammond Philip , Karki Sher , Dang Wei , Edler Collin , Polder Nephi , Chan Cheryl , Jacinto Jonathan , Desjarlais John
Published: April 2020

CD19 is a pan-B cell surface receptor that is expressed from early stages of pre-B cell development through terminal differentiation into plasma cells. It is an attractive immunotherapy target for cancers of lymphoid origin since it is also expressed on the vast majority of Non-Hodgkin Lymphoma (NHL) cells. Although the anti-CD20 monoclonal antibody rituximab is efficacious for the treatment of lymphoid tumors, a growing population of rituximab-refractory patients has resulted in an unmet need for alternative B cell-specific immunotherapeutics. Engineered Fc variants that increase the affinity of IgG antibodies for Fc gamma receptors (FcγR) can dramatically enhance antibody-dependent cell-mediated cytotoxicity (ADCC). This technology has been applied to a humanized anti-CD19 antibody (XmAb™ CD19) and demonstrates ADCC against multiple cell lines representative of follicular lymphoma (FL/DOHH-2), chronic lymphocytic leukemia (CLL/MEC-1), B-cell acute lymphoblastic leukemia (B-ALL/VAL), mantle cell lymphoma (MCL/JeKo-1), hairy cell leukemia (HCL/BONNA-12), and Burkitt’s lymphoma (Raji). The ADCC activity observed is in striking contrast with a wild type IgG1 version of the antibody, which mediates little or no ADCC. Moreover, the XmAb™ CD19 activity is comparable to that of rituximab for the majority of cell lines tested and occasionally superior. Fc variants of this antibody are being evaluated in vitro for their ability to conduct ADCC by various effector cell populations, and in relevant in vivo efficacy models. In summary, these data suggest that anti-CD19 Fc variant antibodies with increased effector function could be promising as NHL immunotherapeutics, particularly for rituximab-refractory NHL.
TRAIL Receptor 1 and TRAIL Receptor 2 Monoclonal Antibodies Induce Apoptosis and Increase Susceptibility to Cytostatic Agents in Mantle Cell Lymphoma.
Authors: Rieken Malte , Pastore Alessandro , Weigert Oliver , Weinkauf Marc , Hutter Grit , Zimmermann Yvonne , Hiddemann Wolfgang , Dreyling Martin
Published: April 2020

Background: TRAIL is a member of the TNF-family of cytokines, which induces apoptosis in various solid tumors by activation of the death receptors DR4 and DR5. Previous studies demonstrated that human agonistic antibodies to TRAIL receptors TRAIL R1 (ETR1) and TRAIL R2 (ETR2) activate the apoptosis pathway via caspase 8. Also, preliminary data suggest an efficacy of the antibodies in human lymphoma cell lines; however, little is known about the effects of ETR1 and 2 in mantle cell lymphoma (MCL), a distinct lymphoma subtype with an especially poor clinical outcome.

Methods: 4 MCL cell lines (HBL2, GRANTA 519, Jeko-1, NCEB-1) and two lymphatic control cell lines (Jurkat, Karpas 422) exposed to different doses of ETR1 and ETR2 were studied for inhibition of proliferation (cell count) and metabolism (WST-1 assay); cell apoptosis was quantified by flow cytometry (Annexin V staining). In addition, all cell lines were also exposed to combinations of ETR1 and various cytostatic compounds (cytarabine, fludarabine or mitoxantrone) to explore potential synergism.

Results: Inhibition of proliferation and induction of apoptosis was achieved in all cell lines in a dose and time dependent manner, but susceptibility varied strongly between the MCL cell lines. With the notable exception of NCEB-1, ETR1 inhibited cell proliferation more effectively than ETR2. IC50 values after 24 hours exposure of ETR1 was 3,26 μg/ml (HBL-2) and 1,09 μg/ml (Jeko-1), whereas no IC50 was reached in the remaining MCL cell lines. Accordingly, HGS-ETR1 at a concentration of 2μg/ml induced apoptosis in 9% (Granta 519) to 63% (Jeko-1) of cells after 24 hours, respectively. In contrast, IC50 values of ETR 2 were only reached in Jeko-1 (0,85μg/ml). Preliminary data indicate a synergistic effect of HGS-ETR1 in combination with conventional cytostatic agents, confirmatory experiments will be presented at the meeting.

Conclusions: Our data indicate that both, ETR1 and ETR2, induce apoptosis in MCL cell lines; however, ETR1 seems to be more effective. Interestingly, combination experiments suggest that induction of apoptosis by TRAIL receptor antibodies may overcome chemotherapy resistance. These results form the rationale for combined approaches in future clinical trials.
Z-Guggulsterone Downregulates Survivin and Induces Cell Death in Large B Cell Lymphoma Cells In Vitro.
Authors: Angelopoulou Maria K. , Lilakos Konstantinos , Salpeas Vassilios , Sachanas Sotirios , Korkolopoulou Penelope , Siakantaris Marina P. , Vassilakopoulos Theodoros P. , Panayiotidis Panayiotis , Pangalis Gerassimos A.
Published: April 2020

Introduction: Survivin is a member of the Inhibitor of Apoptosis Proteins and has recently gained attention as a possible therapeutic target in malignancies, due to its dual role both as an antiapoptotic protein and as a cell cycle regulator. It is overexpressed in malignant cells and confers resistance to chemotherapy and other stimuli triggering apoptosis. Z-Guggulsterone (Z-GGS) is a plant sterol, which has been used in inflammatory conditions and has been recognized as a potent NF-kB suppressor. Since Survivin, as well as other antiapoptotic proteins, are under NFkB regulation, we studied the effect of Z-GGS on two B-cell lymphoma cell lines.

Methods: DB and HT cell lines were treated with increasing concentrations (10μM, 20μM and 30μM) of Z-GGS, for 24, 48 and 72 hours. Survivin expression was tested with Flow Cytometry and Survivin transcripts were measured with quantitative real time PCR using the Universal Probe Library hydrolysis probes and expressed as Survivin/abl ratio. Cell viability was assessed with the MTT assay.

Results: Both cell lines were positive for Survivin at baseline by flow cytometry (66% of total cells for DB and 95% for HT). Treatment of DB cells with 10, 20 and 30μM Z-GGS resulted in a 44%, 49% and 68% reduction of Survivin expression at 24 hours, respectively, whereas the effect on HT was less prominent with a 10% reduction at 24 hours with 30μM Z-GGS. Survivin transcripts decreased as well, with the maximum effect observed at 72 hours with 30μM Z-GGS for both cell lines: Survivin/abl was 0.009 for untreated cells vs 0.0008 with 30μM Z-GGS for DB cells and 0.0135 vs 0.0005 for HT cells. Linearity was observed for increasing concentrations of Z-GGS at 72 hours. Cell viability was practically unaffected at any time point with 10 and 20μM Z-GGS for both cell lines, whereas 30 μM Z-GGS resulted in a 63% and 78% cell death at 48 and 72 hours respectively for DB cells and 67% and 83% for HT cells.

Conclusions: The steroid Z-GGS downregulates Survivin expression in B-lymphoma cells in vitro and induces cell death at 30μM concentration. Further experiments will clarify its possible role in the treatment of B-cell malignancies.
Non Hodgkin Lymphomas Showing Microsatellite Instability (MSI-H NHL) Are Characterized by Both Specific Biological and Clinical Features.
Authors: Borie Claire , Raphael Martine , Buhard Olivier , Duval Alex
Published: April 2020

Cancers showing microsatellite instability (MSI-H) constitute a frequent subset of human tumors characterized by mismatch repair (MMR) deficiency. This phenotype is well known to occur in the Human Non Polyposis Colorectal Cancer (HNPCC) syndrome, as well as in about 10–15% of sporadic colorectal, gastric and endometrial cancers. Of interest, we recently demonstrated that it was also strikingly associated with a subset of Non Hodgkin Lymphomas (NHL) developed in either HIV positive (HIV-RL, e.g. HIV-related Lymphomas) or iatrogenically immunosuppressed (PTLD, e.g. Post-Transplantation Lymphoprolipherative Disorders) patients. We here further characterize this new subset of immunodeficiency-related lymphomas (ID-RL) at both the biological and clinical levels. By using specific markers of the MSI-H phenotype, we extended our molecular screening to a series of 161 NHL developed in either immunocompetent (n = 136) or immunodeficient (n =25, including 4 HIV-RL and 21 PTLD) hosts. Clinically, MSI-H NHL were found to be:

mostly PTLD (12/114, e.g 10%), and more rarely HIV-RL (4/131, e.g. 3%) (p = 0;05; fisher’s exact test): mostly T-cell NHL (4/17, e.g. 24% of T-cell ID-RL, as compared to 10/224, e.g 4% of B-cell ID-RL); late occurring PTLD after organ transplantation associated or not with EBV (52% EBV negative and 48% EBV positive).

Biologically, MSI-H NHL presented with:

primary MMR defects involving MLH1, MSH2 or MSH6; secondary mutational events at target genes for instability, as already reported (RAD-50, BAX, CASP-5 and others…); frequent V599E activating mutation within the BRAF gene (3/9, e.g. 30%); stable cytogenetic profiles characterized by only few chromosomal rearrangements if any.

Taken together, these data show that oncogenic mechanisms involved in MSI-H NHL differ from those already known to be associated with other ID-RL, e.g. Chromosomal Instability or EBV-related pathways, and that they should now be considered as a distinct tumor entity.
Down-Regulation of Bax and Bak in Immuno-Chemotherapy Resistant Non-Hodgkin’s Lymphoma Cells.
Authors: Olejniczak Scott H. , Clements James L. , Bangia Naveen , Hernandez-Ilizaliturri Francisco J. , Czuczman Myron S.
Published: April 2020

The balance between pro-apoptotic (Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL, Mcl-1) Bcl-2 family proteins is essential for the maintenance of B-cell homeostasis. Disruption of this critical balance occurs in the majority of B-cell neoplasms. Clinically, high Bcl-2/Bak and Bcl-2/Bax ratios have been associated with decreased median survival (7.3 years and 3.8 years, respectively) in follicular lymphoma patients1. Recent work in mouse embryonic fibroblasts deficient for the multi-domain pro-apoptotic Bcl-2 family proteins Bax and Bak has shown that they are essential for induction of cell death following apoptotic stimuli that act through the mitochondrial (intrinsic) pathway2. The vast majority of clinically available anti-neoplastic agents, including rituximab, are known to induce cell death via this pathway and therefore likely rely on Bax and/or Bak to exert their anti-tumor effects. Alteration in expression of Bax and/or Bak could therefore underlie acquired resistance to rituximab and chemotherapy in NHL patients. To study the phenomenon of rituximab resistance we developed several rituximab-resistant cell lines (RRCL) that we subsequently showed were also resistant to chemotherapy. RRCL were generated by exposing Raji, SU-DHL-4 and RL cells to escalating doses of rituximab +/− human serum and subsequently cloning by limited dilution. In our present work we studied the efficacy of clinically-applicable chemotherapeutic agents against RRCL. Additionally we studied the intrinsic apoptotic pathway in an attempt to explain shared mechanisms of resistance to chemotherapy and rituximab. We found that RRCL have dramatically reduced levels of both Bax and Bak proteins by Western blot while levels of Bcl-2, Bcl-xL and Mcl-1 protein were comparable to parental cells. Transfection of RRCL with Bax or Bak sensitized them to apoptotic cell death. Currently, we are attempting to validate previous studies that have shown that down-regulation of Bax and/or Bak correlates with a poor prognosis in NHL. Additionally, we are exploring the mechanism(s) by which Bax and Bak are down-regulated in RRCL and primary patient samples.
FOXP3 Expression in Diverse T-Cell Lymphomas.
Authors: Beltrán-Gárate Brady E. , Quiñones Pilar , Morales Domingo , Huamani-Zavala Julia , Mejía Osbert , Vidurrizaga Miriam , Malaga Jose , Castillo-Aguirre Jorge , Riva Luis , de Mendoza Fernando Hurtado , Carrasco-Yalan Antonio A.
Published: April 2020

BACKGROUND: Foxp3 is a key regulatory gene required for the development and function of:

regulatory CD4+CD25high T cells (Treg) specialized in maintaining the balance between immunity and tolerance and activated conventional CD4+CD25low T cells without suppressive activity.

Until now it is not yet possible to study human FOXP3+ Treg irrespective of their CD25 expression. Previous studies had reported FOXP3+ T cells in Adult T-cell Leukemia/Lymphoma cells (ATLL) related to HTLV-1 and in other lymphomas types the FOXP3 expression was only detected in the reactive T-cell background.

AIM: To determine the specifity and prognostic value of the FOXP3 expression in T-cell lymphomas.

METHODS: A retrospective study was performed on 46 samples collected from diverse T-cell lymphomas in our institution. A highly sensitive immunohistochemical method was used to demonstrate FOXP3 protein expression with a mouse monoclonal antibody (clone 236A/E7ABCAM) in most formalin-fixed paraffin-embedded tissue sections from lymph nodes, skin, bone marrow and extranodal sites samples as cavum and stomach. We did not co-stained with CD25 and considered a FOXP3+ tissue when positivity was > 80% of toumor cells. Correlation with survival was done.

RESULTS: Among the 46 evaluable T-cell lymphomas collected, 33 were ATLL, 7 mycosis fungoids (MF) and 6 unspecified peripheral T-cell lymphomas (U-PTCL). Among the 33 ATLL: lymphomatous=17, acute=11, smoldering=1, chronic=1, cutaneous=1 and undefined=2. FOXP3 expression in tumour cells was detected in 24% (8/33) of ATLL cases, was negative in MF tumour cells and detected in 33% (2/6) of U-PTCL. Interestingly FOXP3 expression between 30–40% was expressed in 3 MF cases associated to Treg cells. Among the ATLL cases FOXP3 positivity were obtained in 35% (6/17) of lymphomatous type; 18% (2/11) of acute ones and none in others ATLL types studied. We failed to demonstrated any correlation between FOXP3 status and survival.

CONCLUSIONS: FOXP3 is expressed in ATLL and T-cells peripheral lymphoma. Treg presence in the tumour environment plays an important immune system response role and its identification should be fundamental.
Hemin Confers Resistance to Imatinib (Glivec)-Induced Cell Death but Enhances Nilotinib (AMN107) Cytotoxicity in Human K-562 CML Leukemia Cells.
Authors: Bonovolias Ioannis D. , Manley Paul W. , Tsiftsoglou Asterios S.
Published: April 2020

Imatinib (Glivec) and Nilotinib (AMN107) are selective inhibitors of the Bcr-Abl, Kit and PDGFR tyrosine kinases, developed by Novartis Pharma AG to treat CML and other malignancies by promoting apoptosis (Manley P.W., et al. Biochim Biophys Acta 1754(1–2):3–13, 2005). Heme (iron protoporphyrin-IX) and its oxidized form Hemin, are key regulators of key cellular functions in mammalian cells and Hemin protects normal and leukemia cells from cytotoxic agents (Tsiftsoglou A.S. et al., Pharmacology & Therapeutics 111(2):327–345, 2006). We have recently observed that Hemin, like Erythropoietin (EPO), protects human Ph+ K-562 CML leukemia cells from Imatinib-induced cell death. We extended this observation and investigated whether Hemin also counteracts Nilotinib-induced cytotoxicity in cells. K-562 CML leukemia cells were treated (from 0h to 96h) separately with each of the inhibitors added at different concentrations (0.01 μM to 1 μM) in the presence and absence of Hemin (90 μM) and assessed for their ability to proliferate, differentiate into erythroid lineage or undergo cell death. K-562 cells pre-treated with Hemin prior to be being exposed to Imatinib and/or Nilotinib were used to demonstrate whether induction of differentiation by Hemin alters their developmental stage and susceptibility to either inhibitor. Our results thus indicate that:

Hemin confers resistance to Imatinib in a substantial proportion of cells (20–30%). Priming cells with Hemin gradually decreased susceptibility to Imatinib-induced cell death. Considering that the intracellular Heme in CML cells may overcome the Imatinib effect, this resistance could affect the clinical outcome of CML patients treated with Imatinib. Conversely, Hemin enhanced cell death induced by Nilotinib in the concentration range 0.01 μM to 1 μM.

These findings indicate that Imatinib and Nilotinib induce cell death in K-562 CML leukemia cells, via pathways affected differently by Hemin. Experiments are now in progress to delineate the mechanisms via which Hemin exerts such disparate action (antagonistic and synergistic) on these inhibitors in human leukemias.
Imatinib High Dose (800 mg): Results of a Phase II Trial of the GIMEMA (Gruppo Italiano Malattie Ematologiche Dell’Adulto) CML Working Party in Intermediate Sokal Risk Patients and Status-of-the-Art of an Ongoing Multinational, Prospective Randomized Trial of Imatinib Standard Dose (400 mg Daily) vs High Dose (800 mg Daily) in High Sokal Risk Patients.
Authors: Rosti Gianantonio , Castagnetti Fausto , Amabile Marilina , Testoni Nicoletta , Poerio Angela , Marzocchi Giulia , Breccia Massimo , Palandri Francesca , Intermesoli Tamara , Stagno Fabio , Specchia Giorgina , Abruzzese Elisabetta , Giannoulia Panagiota , Saglio Giuseppe , Pane Fabrizio , Rancati Francesca , Nagler Arnon , Haznedaroglu Ibrahim , PorkKa Kimmo , Hjorth-Hansen Henrik , Nielsen Johan L. , Simonsson Bengt , Martinelli Giovanni , Baccarani Michele
Published: April 2020

Imatinib has become the treatment of choice for CML. The standard dose (SD) for CP CML is 400 mg daily: results are less favourable in pts at high or intermediate Sokal risk vs low Sokal risk ones. In intermediate Sokal risk, the IRIS trial (Hughes et al NEJM 349:15, 2003 ) reported at 12 mos a complete cytogenetic response (CCgR- 0% Ph-pos) rate of 67% and a major molecular response (MMolR) rate of 45%. Pre-clinical and clinical data suggest that high doses (HD - 800 mg daily) of ima may be more effective. The GIMEMA CML Working party is conducting a phase II, multi-istitutional prospective study (serial n. CML/021) to investigate the effects of imatinib HD in intermediate Sokal risk. Between Jan, 2004 and May, 2005, 25 centers enrolled 82 pts (80 eval); median age 56 yrs (26–79). Pts evaluable at 3,6 and 12 mos are 80, 77 and 65, respectively. The median observation time is 12 mos. At 3 and 6 mos, 83% and 97% of the pts reached a stable CHR. At 6 mos, 86% obtained a CCgR and 53% of CCgR pts a MMolR (Bcr-Abl/Abl × 100 ratio < 0.1%). At 12 mos, the CCgR rate was 90% and the MMolR rate was 57%. One patient progressed to accelerated/blastic phase. The compliance to HD treatment was good: at 3, 6 and 12 mos 55%, 52% and 52% of the pts received a median daily dose of imatinib > 600 mg. Non hematopoietic AEs accounted for the great majority of dose reductions. The results of this trial further indicate that imatinib HD induces higher and more rapid responses in intermediate Sokal risk CML pts in early chronic phase, being superior to the results obtained with SD (IRIS) and in the range of the MD Anderson results (Kantarjian et al Blood 2004 103:2873). A second project is reserved to high Sokal risk CML pts in early CP: a multinational group, within EuropeanLeukemianet CML WP, is conducting a phase III trial (1:1) of imatinib 400 mg vs 800 mg. By July 31, 2005, 141 patients have been enrolled: GIMEMA (88 pts), Nordic CML Study Group (Sweden, Denmark, Norway and Finland) (25 pts), Turkey (25 pts) and Israel (3 pt).
Synergistic Activity of Nilotinib and Established Chemotherapeutic Agents in Imatinib-Sensitive and -Resistant BCR-ABL-Positive Leukemia Cells.
Authors: Radujkovic Aleksandar , Topaly Julian , Ho Anthony D. , Zeller W.J. , Fruehauf Stefan
Published: April 2020

Resistance towards the tyrosine kinase inhibitor (TK) imatinib frequently emerges in advanced stages of chronic myelogenous leukemia (CML) and complicates disease treatment. Nilotinib (AMN107) represents a novel BCR-ABL TK inhibitor with reported antiproliferative activity against imatinib-resistant cells in vitro. Since combination chemotherapy is a strategy to increase the antileukemic efficacy, we were interested whether combinations of nilotinib and other established chemotherapeutic agents such as mafosfamide, busulfan, treosulfan, daunorubicin or mitoxantrone display synergistic activity in different imatinib-sensitive and -resistant BCR-ABL-positive CML cells. Tetrazolium-based MTT assays (48 h incubation) revealed as expected that substantially higher concentrations of single agent nilotinib are required for growth inhibition of the imatinib-resistant K562-R (IC50=19.76±4.94 μM) and LAMA84-R (IC50=0.14±0.03 μM) cell lines as compared to the imatinib-sensitive counterparts (IC50=0.10±0.03 μM for K562 and IC50=0.03±0.01 μM for LAMA84, respectively). Combination treatment of both imatinib-sensitive LAMA84 and -resistant LAMA84-R cells resulted in variable antiproliferative effects. Combination index (CI) values calculated according to the median-effect method of Chou and Talalay denoted mostly additive (CI=1) and antagonistic (CI>1) effects. In both lines a clear synergistic activity with CI<1 could be observed only for the combination nilotinib plus mitoxantrone. By comparison in the imatinib-sensitive K562 and -resistant K562-R lines all applied combinations showed improved antileukemic efficacy with CI values predominantly lower than 1 indicating synergism between nilotinib and most of the substances investigated (see Table I, n=3, results are given as mean CI values at different growth inhibition levels (fraction affected, Fa)).

In BaF3 cells expressing imatinib-resistant mutants of BCR-ABL (E255K and T315I) IC50-values of single agent nilotinib were 0.37±0.04 μM and 23.58±1.92 μM, respectively. Importantly, combination treatment of imatinib-resistant BaF3 cells with both nilotinib plus daunorubicin and nilotinib plus mitoxantrone led to enhanced antiproliferative effects with synergism (CI<1) at high growth inhibition levels even in the T315I resistant mutant cell line (see Table II, n=3).

The results demonstrate the ability of established chemotherapeutic agents to enhance the antileukemic efficacy of nilotinib and indicate the potential of the applied combinations for further study in the clinical setting.
AMD3100 Inhibits Chemotaxis towards SDF-1 and CXCR4-Mediated Stroma-Contact in a Dose-Dependent Manner, Resulting in Increased Susceptibility to Imatinib.
Authors: Dillmann Falk M. , Veldwijk Marlon R. , Calandra Gary , Zeller Werner J. , Ho Anthony D. , Fruehauf Stefan
Published: April 2020

Besides new efforts in the treatment with tyrosine-kinase-inhibitors like imatinib were made, Chronic myelogenous leukemia (CML) still remains chronic with a progression of the disease by time. New ways of treatment have to be found to eradicate the malignant clone in the bone marrow.

In our study we are evaluating whether an inhibition of the SDF-1-CXCR4-axis by the selective CXCR4 receptor antagonist AMD3100 results in increased cell kill of the BCR-ABL+ cell line BV-173 when combined with treatment with imatinib. AMD3100 significantly blocks binding of the FACS-anti-CXCR4-Mab to its corresponding receptor. Furthermore, AMD3100 significantly inhibits SDF-1 mediated chemotaxis towards 500ng/ml SDF-1 to 0.71±0.15 of spontaneous transmigration at the highest concentration (100μM) of AMD3100 whilst SDF-1 500ng/ml leads to 1.4±0.06 fold spontaneous transmigration (n=3). Even lower concentrations (10μM and 1μM) still significantly reduce transmigration to a level lower than spontaneous transmigration. Also migration into the SDF-1 producing stromal-cell line FBMD-1 was dose-dependently significantly reduced to 56.7% ± 8.6% migration of untreated controls at 100μM AMD3100 (n=4). Lower concentrations also significantly reduced migratory activity of BV-173. In a stroma-dependent co-culture AMD3100 dose-dependently increased susceptibility of BV-173 towards simultaneous treatment with Imatinib at a dose of 1.6μM. We saw 23% ± 8% PI+ cells in the control without AMD3100 vs. 40% ± 12% PI+ cells al 100μM AMD3100 (n=3; p=0.06). Incubation with 10μM and 1μM AMD3100 only resulted in a slight increase of PI+ cells to 28% ± 13% at 1μM and 29% ± 3 at 10μM respectively.

The same assays on the AML-cell line HL-60 and on the BCR-ABL+ cell line SD-1 led to a significant reduction in SDF-1 dependent chemotaxis and stromal-dependent migration especially at the 100μM concentration but on a level with higher transmigration rates. Interestingly, for these cell lines we found lower CXCR4 surface expression (MFI: 478 ± 71 for the BV-173 vs. 29 ± 8 for the HL-60 and 17 ± 1.5 for the SD-1) going hand in hand with lower effects on migration rates following incubation with AMD3100 than we could show for the CML cell line BV-173.

We conclude that co-treatment with AMD3100 and Imatinib leads to a sensitization of BCR-ABL+ leukaemia cells which is dependent on the baseline CXCR4 expression. This is a promising strategy to reduce residual BCR-ABL+ leukaemia cell load in CML patients.
Philadelphia Chromosome (Ph’) Secondary Clones in Chronic Myeloid Leukemia (CML) Are Not Indicative of Resistance to IFNa- or Imatinib-Based Treatment Regimens.
Authors: Falchi Lorenzo , Prontera Paolo , Luzi Debora , Festuccia Viola , Emili Rita , Gottardi Enrico , Donti Emilio , Liberati Anna Marina
Published: April 2020

The rate of complete karyotypic remission (CKR) and complete molecular remission (undetectable BCR/ABL specific transcript by real-time quantitative-PCR (Gabert J et al. Leukemia: 2003; 17: 2318–57) and NESTED/real-time-PCR (Guo JQ et al.; Leukemia: 2002; 15:2447–53)) to various therapies was analysed in 35, of the 114 CML patients observed in our institution, who showed cytogenetic clonal evolution. Overall, 52 karyotypic abnormalities additional to Ph’ were documented at the time of diagnosis (early clonal evolution) or late in the course of the disease (late clonal evolution). Seven of 12 patients with early clonal evolution achieved one (5 pts) or more (2 pts) CKRs, for a total number of 9, in response to IFNα (2 CKRs), IFNα plus ARA-C (1 CKR), imatinib (5 CKRs) or imatinib-IFNα combination (1 CKRs). Six of the 23 patients with late clonal evolution achieved one or more CKRs during the course of their disease after treatment with IFNα (2), IFNa plus ARA-C (1) or imatinib (5). In addition, 7 of the 13 CK responders reached a complete molecular remission in response to IFNa (1), IFNa plus ARA-C (1) imatinib (1), or imatinib plus IFNa (4). The 5 complete molecular remissions documented in patients with early clonal evolution, were observed following treatment with IFNα plus ARA-C (1), imatinib (1), or imatinib plus IFNα (3). Two patients with late clonal evolution obtained complete molecular remission in response to IFNα and imatinib plus IFN therapy, respectively. Cytogenetic and molecular response rates were higher in patients with early (58%, 57%) than in patients with late secondary clones (26%, 33%), while there were no significant differences in time to achieve cytogenetic (2–20 months vs 1–25 months) or molecular (3–26 months vs 13–52 months) remission and the duration of response (11 months, range 4–42 vs 10 months, range 7–46 and 2–19 vs 3–13+ months, respectively) between the two groups. Finally, no relationship was evident between the type of additional karyotypic anomalies and sensitivity to treatment. In fact, clones bearing complex translocations (2), isoPh’ (2), trisomy 8 (2), translocations other than Ph’ (2), partial chromosome monosomy (11-q, 1) or loss of chromosome Y alone (1) or associated with other anomalies (−6 and −14 or −21 or isoPh’) (3) all responded to therapy. In conclusion, although the small sample size, these findings suggest that the presence of karyotypic abnormalities additional to Ph’ in CML is not associated to disease resistance to therapy.
Loss of JAK2 in CML Cell Lines.
Authors: Vaughan Beverly , Howard Julie , Brazma Diana , Nacheva Elisabeth
Published: April 2020

Chronic Myeloproiferative Disorders (CMPDs) are a spectrum of haematological malignancies of which the molecular pathogenesis remains unknown. Chronic Myeloid Leukaemia (CML) being the only disorder with a recognisable pathogenomic abnormality, the Ph chromosome. Recent published data has supported the role of tyrosine kinases in the disease pathogenesis, in particular the role of a cytoplasmic tyrosine kinase JAK2. It has been shown that >80% of Polycythaemia Vera (PV) patients and 30% of Essential Thrombocythaemia (ET) patients demonstrate a clonal and recurrent mutation V617F in the JH2 pseudo-kinase domain of JAK2 located on 9p24.1. Our array CGH investigations of cell lines, established from lymphoid and myeloid blast phase samples revealed a complex pattern of imbalances affecting the short arm of chromosome 9: on the background of 2–3-fold amplification, a loss of the 9p24.1 region, which harbours the JAK 2 sequences was repeatedly observed. FISH using BAC DNA probes from the 9p24.2 and 9p23 region was carried out to map the JAK2 gene and the flanking sequences. Results so far indicate the presence of both imbalances (amplifications & deletions) and rearrangements involving the JAK2 region in 5 cell lines. An on going FISH study of CMPD patients (CML, PV, ET and MF) with normal and abnormal karyotypes has revealed 9p24 imbalances in 18% (5 out of 28). The observation of JAK2 aberrations in a range of haematological malignancies demonstrates the potential importance of tyrosine kinases in the leukemogenesis. The pathegenomic significance of aberrations found in the region surrounding JAK2 has yet to be defined and our study demonstrates the importance of molecular cytogenetics as a tool in understanding the significance of JAK2 in CMPDs.
Point Mutation and Alternative Splicing in the ABL Kinase Domain as a Mechanism for Imatinib Resistance.
Authors: Chen Rong , Potts Steven , Ma Wanlong , Kantarjian Hagop , Giles Francis , Albitar Maher
Published: April 2020

Missense point mutations in the region encoding the ABL tyrosine kinase domain have been reported in approximately 35% of patients with imatinib-resistant chronic myeloid leukemia (CML). The reported mutations result in reactivation of the BCR-ABL tyrosine kinase. Screening patients with imatinib-resistant CML, we identified 42 different mutations in the ABL tyrosine kinase domain-encoding region, 2 of which were silent (no amino acid change): A864G and G909A. The A864G mutation was associated with a 54-nucleotide reduction in the length of the mRNA transcript, representing a loss of nucleotides 1089-1143; the G909A mutation was associated with a normal-length transcript. The nt1089-1143 transcript deletion represents a partial exon deletion in which the first half of exon 8 is skipped, suggesting that A864G leads to abnormal splicing. Splicing is regulated by 6- to 8-nucleotide exonic splicing enhancer (ESE) and exonic splicing silencer (ESS) motifs recognized by the SR proteins (a family of splicing factors). We therefore used ESEfinder to examine whether A864G or G909A alter ESE motifs, which could block the ability of SR proteins to recognize and bind. This search showed that A864G is at the 7th position of an AGCTGCAG ESE motif, a binding site for SR35, and is within 35 bp of the intron-exon junction. In total, ESEfinder predicted 18 putative SR35-binding ESEs within 50 bp of the intron-exon junction, covering 20% of the kinase domain. The AGCTGCAG motif is conserved in primates (chimpanzees and monkeys) but not in rodents, while A864G is found in mice Although similar links cannot be made with the G909A mutation, these data suggest that imatinib resistance may develop in some patients through alternative splicing and the expression of a truncated (or potentially elongated) protein.
Cas-L Suppresses Progression of Leukemia Induced by p210Bcr/Abl.
Authors: Seo Sachiko , Nakamoto Tetsuya , Suzuki Takahiro , Takeshita Masataka , Ichikawa Motoshi , Chiba Shigeru , Ogawa Seishi , Honda Hiroaki , Oda Hideaki , Kurokawa Mineo
Published: April 2020

Cas-L (Crk-associated substrate lymphocyte type) which is also known as Hef1/Nedd9 was first identified as a protein tyrosine-phosphorylated upon stimulation of β1 integrin in peripheral T cells. Cas-L is a member of Cas family proteins, with a single Src homology (SH) 3 domain and multiple YXXP motifs (substrate domain) and functions as a docking protein in focal adhesions. Bcr/Abl is a fusion oncoprotein that induces chronic myelogeneous leukemia and acute lymphoblastic leukemia. Previous studies reported that Cas-L is tyrosine-phosphorylated by p190Bcr/Abl in lymphoid cells. In this study, we found that Cas-L is also expressed in myeloid cells, which suggests functional interaction between CasL and p210Bcr/Abl. To elucidate the role of Cas-L in leukemia induced by p210Bcr/Abl, we generated the Cas-L-deficient p210Bcr/Abl transgenic mice. The mice displayed early development of myeloproliferative disease compared with the p210Bcr/Abl transgenic mice, which results in the early death of those mice. Pathologically, increased infiltration of myeloid cells into several tissues was detected in the absence of Cas-L. No significant differences were detected in the proliferation assay using bone marrow cells from those mice. Taken together, Cas-L seems to negatively regulate the progression of p210Bcr/Abl-induced leukemia presumably by inhibition of extramedullary hematopoiesis.
Chronic Myeloid Leukemia (CML) Treatment in Brazil: Results of the Rio Grande do Sul Cooperative Group for Treatment of CML.
Authors: Fogliatto Laura M. , Guterres Ines , Capra Marcelo Z. , Fernandes Mario S. , Schaan Marisa , Hellwig Tania , Zago Aletea , Schilling Marco , Almeida Denise , Wisinteiner Sabrina , Lorandi Roque , Winck Adriana , Zardo Adriana , Silla Lucia M.R.
Published: April 2020

Background: Imatinib mesylate (Glivec,Novartis), an inhibitor of Bcr-abl tyrosine kinase, is currently being used in the treatment of CML with hematological, cytogenetic and molecular response. In Brazil, this drug is approved by Health Department for chronic phase (CP) as a second line drug and as first line for accelerated (AP) and blastic phase (BP). Rio Grande do Sul (RS) Cooperative Group for Treatment of CML coordinates the treatment of this disease in Rio Grande do Sul, the southern most state of Brazil that differs from the north part of the country regarding climate, population profile (mostly European descendents) and a high number of farm workers.

Methods: 211 CML Ph+ patients are presently being treated in RS. Sex distribution: 96 male and 115 females, aged 11–80 years (median 48 y);166/211 in CP, 42/211 in AP and 11/211 in BP. Imatinib daily dose is 400 mg (CP) and 600 mg (AP and BP). 166/211 patients are in treatment with imatinib and 32/211, interferon or hydroxyureia. In imatinib group, 144/166 patients are in complete hematologic response and 57/166 in complete cytogenetic remission.

Conclusions: in Brazil, imatinib treatment is limited by economic problems and the drug is used latter in course of disease. This may compromise the results, as seen in the low cytogenetic complete response described above. We are now investigating this results in order tofind other related factors.
Rituximab Is the Essential Treatment Modality That Underlies the Significant Improvement in Short and Long Term Outcome of Patients with Advanced Stage Follicular Lymphoma - A 10 Year Analysis of GLSG Trials.
Authors: Hiddemann Wolfgang , Hoster Eva , Buske Christian , Dreyling Martin , Kneba Michael , Hallek Michael , Lengfelder Eva , Wandt Hannes , Unterhalt Michael
Published: April 2020

Recent reports have shown a significant improvement of overall survival (OS) in patients with advanced stage follicular lymphoma (FL) over the last few decades. In order to identify the essential treatment modalities that underlie these observations two consecutive study generations of the German Low Grade Lymphoma Study Group (GLSG) covering a ten year period were analyzed for remission rates (RR), time to treatment failure (TTF) and OS. The analysis was adjusted for risk factors using the FLIPI, and type of primary induction therapy (including Rituximab or not). Between 1996 and 2005 complete data sets of 1332 patients with stage III or IV FL who were in need of therapy were collected. Therapy comprised prospective randomized comparisons of CHOP versus MCP in GLSG study ’96 (1996 – 2000) and CHOP versus R-CHOP in GLSG study ’00 (1996 – 2005). The entry criteria and the distribution of risk factors were identical in both study generations. The type of chemotherapy had limited impact on RR with CHOP being superior to MCP (86% versus 77%, p=0.0094) but no impact on TTF or OS. Patients treated in GLSG ’00 had a significantly better RR (94% versus 88%, odds ratio 0.47, p=0.0002), a longer TTF (median 48 months versus 32 months, relative risk 0.66, p<0.0001) and a longer OS (89% versus 78% at four years, relative risk 0.42, p<0.0001) as compared to patients of GLSG ’96. Multiple Cox regression analysis revealed Rituximab as being the determinant responsible for the differences in RR (adjusted odds ratio 0.45, p<0.0001) and TTF (adjusted relative risk 0.39, p<0.0001). With regard to OS, after adjusting for Rituximab as most relevant determinant (adjusted relative risk 0.44, p = 0.0064), the difference between the study generations still remained significant (adjusted relative risk 0.57, p = 0.0071). Hence, Rituximab is the essential treatment modality that underlies the improvement in short and long term outcome of patients with advanced stage FL. Most probably, more effective salvage therapy with Rituximab containing combinations account also for the additional improvement in OS.
Comparison of Erythroid Response (ER) Rates in Myelodysplastic Syndromes (MDS) Patients Treated with Different Therapeutic Strategies Using Epoetin Alfa (EPO).
Authors: Moyo Victor , Lefebvre Patrick , Bourezak Ahmed , Yektashenas Behin , Mundle Suneel
Published: April 2020

Background: EPO continues to be a primary therapeutic choice for the treatment of anemic patients with low to intermediate-1 risk MDS. Initially, without standardized response criteria, ER rates were modest. Recently, a meta-analysis of EPO monotherapy studies in MDS demonstrated a significant improvement in ER rates with the application of standardized International Working Group criteria (IWGc) for response evaluation (Moyo V et al. 2006 ASCO Annual Meeting Proceedings Part I. Vol 24, No. 18S (June 20 Supplement), 2006: 6572). This meta-analysis was expanded to determine the impact of higher doses of EPO or the addition of granulocyte or granulocyte-macrophage colony stimulating factor (G- or GM-CSF) to EPO on ER rates (defined as major + minor response).

Methods: A systematic review and data extraction of studies from PubMed, ASCO and ASH proceedings from 1990–2006 in MDS patients treated with EPO monotherapy or EPO + G-/GM-CSF combination therapy was performed. Pooled estimates of ER rates were calculated in studies defining ER rates without IWGc application (non-IWGc) for EPO monotherapy at Standard (std) dose (30,000–40,000 Units per week) in comparison to studies that defined ER rates using IWGc, where EPO was used at std dose or high dose (60,000–80,000 Units per week) or in combination with G-/GM-CSF.

Results: A total of 23 studies were evaluated in this analysis. As shown in the table below the IWGc studies using EPO at std dose or high dose or in combination with G-/GM-CSF demonstrated significantly higher ER rates than the non-IWGc studies (p<0.001 for all comparisons). Among the IWGc studies, those using a higher dose of EPO alone had significantly higher ER rates as compared to studies using std dose EPO monotherapy (p<0.001) or EPO + G-/GM-CSF combination (p=0.018). In addition, among the studies using EPO combinations, those using EPO + G-CSF (Total studies-3, evaluable N= 96) showed ER rates of 55.2% as compared to 42.8% (p=0.142) for the EPO + GM-CSF combination (Total studies-3, evaluable N= 56).

Conclusions: The findings from this present meta-analysis confirm the impact of IWGc on ER rates for EPO therapy previously observed. Further improvement in ER rates was observed with the use of higher doses of EPO alone or the combination of EPO + G-/GM-CSF. Increasing the EPO dose appeared to have a greater impact on ER than the addition of G-/GM-CSF.

Non-IWGc IWGc Study Group EPO Std Dose EPO Std Dose EPO High dose EPO + G-/GM-CSF Starting EPO Dose Units/week 30,000–40,000 30,000–40,000 60,000–80,000 30,000–40,000 No. of Studies 8 5 4 6 No.of Evaluable Patients 262 393 196 152 ER Rates (%)(95% CI) 32.1 (26–38) 47.8 (43–53) 63.3 (57–70) 50.7 (42–59)
Familial Myelodysplastic Syndromes with Centromere Instability of Chromosome 1 Origin.
Authors: Raaschou-Jensen Klas , Andersen Mette Klarskov , Nielsen Bendt , Kjeldsen Eigil , Kjeldsen Lars
Published: April 2020

Myelodysplastic syndrome (MDS) is a highly heterogeneous disease. The etiology involves both environmental and genetic factors. Identification of new pathogenetic mechanisms may have important impact on the choice of treatment as well as the development of new treatment options. Familial disease gives a unique opportunity to study those mechanisms. Familial occurrence of MDS is rare and mainly associated with autosomal recessive genetic syndromes such as Fanconi anaemia, Bloom syndrome, Diamond-Blackfan syndrome and megakaryocytic thrombocytopenia. Cytogenetic abnormalities are in general seen in 50% of MDS cases. Most frequently, abnormalities involving chromosome 5 or 7 are observed, which has also been described in rare cases of familial MDS. We here present a unique family in which a father and two of his three children were diagnosed with refractory anemia (RA) with cytogenetic aberrations resulting in three different translocations involving chromosome 1q and resulting in trisomy of 1q. Trisomy 1q is often seen in sporadic MDS, but the unbalanced translocations observed in this family may originate from inherited centromere instability of chromosome 1. The genetic background for the observed centromere instability could be a DNA methylation deficiency, which we plan to investigate further.