bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor Necrosis Factor Alpha Inhibits Telomerase and Induces Senescence in Acute Myeloid Leukemia Cells.
Authors: Beyne-Rauzy Odile , Recher Christian , Dastugue Nicole , Prade-Houdellier Nais , Pottier Geraldine , Demur Cecile , Sabatier Laure , Laurent Guy , Mansat-DeMas Veronique
Published: April 2020

Tumor necrosis factor alpha (TNFα) is a multifunctional cytokine, which is overproduced in a series of hematological diseases, including acute myeloblastic leukemia (AML) and myelodysplasia (MDS). Apoptotic cell killing has been the most frequently documented cellular effect of TNFα in AML cells. Much little is known about TNFα effect in AML cells, which display an intrinsic defect in their apoptotic machinery. In this study, we describe that prolonged exposure to TNFα, but not Fas, resulted in senescence in the immature CD34+ CD38− KG1 cell line. Indeed, TNFα-treated KG1 cells displayed growth arrest, increased senescence-associated β-galactosidase activity, telomere shortening and additional chromosomal abberations. In these cells, as well as in fresh AML cells, treatment with TNFα resulted in decreased hTERT gene trancription and hTERT activity. Moreover, we found that ceramide production and JNK activation were critical signaling components in TNFα-induced hTERT gene inhibition. GM-CSF stimulation inhibited ceramide production, JNK activation, hTERT inhibition, and senescence markers induction. To conclude, our study suggests that, at least in some AML cells, TNFα is a regulator of hTERT with important functional consequences in terms of growth capacity and genetic stability. Moreover, the pro-senescent effect of TNFα is efficiently counter-regulated by stimulatory cytokines such as GM-CSF.
Involvement of Altered PML/RARalpha and RARbeta Expression in Delayed Restoration of Retinoic Acid Sensitivity in UF-1, an Acute Promyelocytic Leukemia Cell Line Carrying a Patient-Derived Mutant PML/RARalpha(Arg611Trp).
Authors: Hoshi Yoshiyuki , Imaizumi Masue , Iinuma Kazuie
Published: April 2020

[Introduction]

Mutations in the PML/retinoic acid receptor alpha (RARa) may be one mechanism of acquired resistance to all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APL), but the molecular pathogenesis of this remains unclear. Recently, we reported the delayed restoration of ATRA sensitivity in UF-1, an APL cell line carrying a patient-derived mutant PML/RARa (Leukemia Res, 2004). In this study, we investigated the roles of the mutant PML/RARa and normal RARa in the restored ATRA sensitivity of UF-1.

[Methods]

After culture in the presence of ATRA or Am80 (RARa-selective RA), the differentiation and apoptosis of UF-1 were evaluated using FACS. RA-dependent transcriptional activity was examined using the luciferase assay. The expression of PML/RARa, RARa, and retinoic acid receptor beta(RARb)was analyzed using RT-PCR and Western-blotting.

[Results]

UF-1 had little response to ATRA up to day 4, but subsequently showed differentiation and apoptosis in response to ATRA or Am80 at 1 microM, but not 100 nM or lower. In the presence of Am80, UF-1 underwent differentiation similar to that in response to ATRA, while it showed apoptosis at lower degrees than that induced by ATRA. In the luciferase assay, the mutant PML/RARa had little RA-dependent transcriptional activity, but retained a dominant-negative action on normal RARa. Expression of the chimeric protein was decreased in the presence of 1 microM ATRA after day 4, while RARa was preserved. Interestingly, RARb expression was up-regulated in the presence of 1 microM ATRA.

[Discussion]

The mutant PML/RARa (Arg611Trp), with a reduced ATRA response and a preserved dominant-negative action, caused ATRA resistance of UF-1 during the initial culture period. However, an ATRA-induced decrease in the chimeric protein was thought to be the mechanism by which UF-1 restored ATRA sensitivity after 4 days of culture. Furthermore, the RA signaling that led to apoptosis of UF-1 may be transduced via RARb, which was up-regulated by 1 microM ATRA.
Apoptotic and Multi-Drug Resistance Profile in Elderly AML Patients.
Authors: Vidriales Maria-Belen , Suarez Lilia , García-Laraña José , López Antonio , Moreno María José , Martinez Rafael , Tormo Mar , Lavilla Esperanza , López-Berges Consuelo , de Santiago María , San Miguel Jesús F. , Orfao Alberto ,   for the Pethema Cooperative Group
Published: April 2020

Elderly AML patients have an unfavorable prognosis with frequent resistance of leukemic cells to chemotherapy. The expression of proteins related with apoptosis and multi-drug resistance have been involved in chemotherapeutic resistance in AML; however, these proteins are not only expressed on leukemic but also on normal cells. Moreover, within leukemic cells, different stages of maturation may occur. The aim of the present study was to evaluate the expression of proteins related to apoptosis (APO 2.7, bax, and bcl-2), and drug resistance (P-gp, MRP, LRP) in a series of 117 elderly AML patients (median age of 70) uniformly treated according with the Spanish Pethema cooperative group protocol (LMA >65). 63 patients (54%) achieved complete remission (CR), with a median relapse free survival (RFS) of 9.2 months. In the bone marrow (BM) pre-treatment, we performed a four-color staining technique (APO2.7, bax or bcl-2 with CD32/CD34/CD45; and P-gp, MRP, or LRP with CD34/CD19/CD33), allowing the identification of myeloid blast cells, and the discrimination between stages of maturation of blast cell subsets. Samples were acquired on a FACSCalibur flow cytometer (Becton/Dickinson), and appropriate controls were used in each individual sample and, accordingly, antigen expression in individual cell samples was quantified as relative fluorescence intensity (RFI) (ratio blast cell/control mean channel fluorescence value). Our results showed that the level of expression of MRP and LRP was significantly higher (mean±SD of MFI 2.0±0.75 vs 2.7±1.87 and 4.5±2.2 vs 7.0±6.2, respectively -p <0.03-) in cases that did not achieved CR with one or two cycles of chemotherapy. Although different patterns of expression of apoptosis or drug resistance-associated proteins were detected according with the stage of maturation on myeloid blast cells, we did’t found any other correlation between levels of bcl-2, bax, APO 2.7, bcl-2/bax ratio or MDR and probability of achieving morphological CR. In addition, CD34 expression on blast cells had also significant influence on response (CR was achieved in 81% of CD34 negative cases vs only 46% of CD34 positive cases; p= 0.005). We didn’t found any influence of apoptosis and/or drug-resistance proteins expression in relapse rate nor RFS. Variables influencing RFS were age (p=0.04), and number of cycles to achieve CR (p=0.01); while only WBC count (p=0.05) influenced overall survival (OS). We conclude that MRP and LRP could have a role in the resistance to induction treatment in elderly AML patients but not in RFS or OS.
A Host Cell Reactivation Assay for DNA Repair System Analysis in Primary Hematopoietic Cells.
Authors: Baesecke Joerg , Thoms Kai , Roedling Tina , Aut Birgit , Viereck Volker , Griesinger Frank , Haase Detlef , Neumann Christine , Truemper Lorenz , Emmert Steffen
Published: April 2020

Therapy related acute myelogenous leukemia (t-AML) is frequently observed among patients who undergo a high dose chemotherapy and its incidence varies between 5 to 15% in non-myeloablative and myeloablative treatment protocols. Deficiencies in individual DNA-repair systems seem to be involved in t-AML leukemogenesis since indirect genetic markers of impaired DNA repair, e.g. microsatellite instability (mismatch repair) and polymorphisms of XRCC genes (base/nucleotide excision repair), are frequent in these patients. To directly determine the activity of different DNA repair systems we developed a modified host cell reactivation (HCR) assay which is applicable for the analysis of primary hematopoietic cells of clinical samples. This assay determines the ability of transfected host cells to repair damaged plasmid DNA as reflected in the recovery of luciferase activity. Depending on the type of DNA damage introduced to the plasmid prior to transfection, different DNA repair systems can be analysed, e.g. nucleotide excision repair (NER) or double strand break repair. We performed the HCR assay using 75 to 250 ng luciferase reporter gene plasmid (pCMV-Luc). Lymphocytes and cord blood CD34+-progenitor cells from healthy donors were collected according to the convention of Helsinki. 200,000 to 2,000,000 cells were cryopreserved, thawed and transfected using DEAE-dextran at a concentration of 0,1 mg/ml in a transfection volume of 250 μl. We obtained luciferase activities of 350-fold above background in CD34+ progenitor cells (1000-fold in lymphocytes) rendering these cells applicable for DNA repair analysis. In addition, we evaluated the normalized NER capacity (1000 J/m2 UVC irradiated plasmid vs. unirradiated control) of lymphocytes and two AML cell lineages, Kasumi-1 and HL60. Kasumi-1 and HL60 cells exhibited a significantly reduced NER capacity compared to lymphocytes (6.15% +/− 1.57% and 6.5% +/− 1.59% vs. 12.3% +/− 3.2%). Clinical AML samples are currently been investigated. Our modified HCR can be used for functional DNA repair analysis in fresh and cryopreserved patient samples of pre- and post-leukemic conditions as well as in leukemic blasts to elucidate the role of defective DNA repair during t-AML leukemogenesis. Furthermore, the modified HCR may also be used to determine the individual susceptibility for therapy related myeloid leukemia prior to chemotherapy.
Mimosine Induces Apoptosis through Metal Ion Chelation, Mitochondrial Activation and Reactive Oxygen Species Production in Human Leukemic Cells.
Authors: Hallak Maher , Dvilansky Alexander , Shpilberg Ofer , Levi Itai , Mazar Julia , Nathan Ilana
Published: April 2020

Mimosine, a non-protein amino acid, acts as a reversible inhibitor of DNA replication, and is widely used to synchronize cells at G1 phase of the cell cycle. We tested the possibility that mimosine might have an apoptotic effect on two types of AML cells: the monoblastic U-937 and the promyelocytic HL-60 cell lines. We show that mimosine induces apoptosis in both cell lines, with U-937 cells being more sensitive. The apoptotic effect of mimosine was antagonized by the addition of exogenous iron, indicating that it may act through iron chelation. Its mode of action was thus compared to that of desferrioxamine (DFO), a therapeutic iron chelating agent. Mimosine and DFO differed in their sensitivity to the suppressive effect of exogenous sources of iron in the form of hemin and ferrous sulfate suggesting different targets of action. Addition of another metal ion cupric sulfate was also able to antagonize the apoptotic effect of mimosine, undermining the notion that apoptosis is mediated through inhibition of ribonucleotide reductase, since this enzyme is solely dependent on iron for its activity. Moreover, when higher concentrations of iron were added to mimosine, cell death shifted from apoptosis to necrosis. Induction of apoptosis by both mimosine and DFO caused an early reduction in mitochondrial transmembrane potential and increase in caspase-3 activity, while only mimosine induced oxidative stress. In summary, our results imply that besides its known effect on DNA synthesis and G1 arrest, mimosine also activates apoptosis through an intrinsic pathway as well as reactive oxygen species production and thus elicits its anticancer effect by multiple pathways.
Degree of Concordance between Peripheral Blood Leukemic Blast Count and Mid Induction Bone Marrow in Childhood Acute Lymphoblastic Leukemia.
Authors: Al-Saleh Mahasen M. , Owaidah Tarek , Al-Daama Saad , Al-Humaidan Hind A. , Mousa Emad , Al-Asaad Tarek , Silo Ameurfina , Belgaumi Asim
Published: April 2020

While different Pediatric ALL study groups have used varying definitions of early response (BM vs. PB, prophase vs. day 7 vs. day 14), all agree that it provides critical prognostic information. Bone Marrow aspiration and biopsy (BMA/B) is an invasive procedure requiring sedation or anesthesia, and an early/mid induction specimen may be difficult to interpret even by experienced hematopathologists. In this study we attempted to determine if there was a concordance between peripheral blood blast (PBB) clearance and the findings of the day 14 BMA/B, and whether day 7 PBB count could reliably replace a mid induction BMA/B. Clinical data for newly diagnosed pediatric (<14 years) ALL patients between January 1999 and December 2001 were retrieved from our prospective database. Day 14 BMA/B slides were reviewed independently by two hematopathologist. For the total 165 patients, median age was 4 years, 53.9% were boys. Complete information was available for 151 of these patients and further analysis is based on this number. 124 (82.1%) were treated with 4 agents while the remainder received a 3-drug induction. 23 (18.5%) had positive PBB on D7, and 21 (13.9%) had >5% blasts in the D14 BMA/B. The D7 PBB count could positively and negatively predict the D14 BMA/B 71.9% and 89.4% of the times, respectively. In conclusion, when the D14 BMA/B is used as a measure of early response, an absence of D7 PBB can reliably predict a negative BM, however persistence of PBB does not necessarily predict a sub-optimal BM response to early therapy. Therefore, patients without PBB on D7 may not require BMA/B on D14, therefore avoiding an invasive procedure for this group of patients.
Scutellaria Baicalensis, a Herbal Medicine: Antitumor Activity Against Acute Lymphocytic Leukemia, Lymphoma and Myeloma in Vitro.
Authors: Kumagai Takashi , Muller Claudia , Desmond Julian C. , Imai Yasufumi , Heber David , Koeffler H. Phillip
Published: April 2020

Scutellaria baicalensis is a widely used Chinese herbal medicine for anti-inflammatory and anti-cancer therapy. The anticancer activity of Scutellaria baicalensis against prostate, breast, hepatocellular, colon and squamous cell carcinomas in vitro has been reported. In this study, we initially investigated its in vitro antitumor activities against 29 cancer cell lines including prostate, breast, ovarian, endometrial and pancreatic cancers as well as myeloid and lymphocytic leukemias, lymphomas, myelomas. The cells were cultured with Scutellaria baicalensis at a dose of 50 μg/ml for 4 days. The herb showed strong anti-proliferative activities against Blin-1 and Nalm-6 acute lymphocytic leukemia cells, Daudi Burkitt′s lymphoma cells and NCI-H929 myeloma cells as measured by MTT assays. Dose-response studies showed an ED50 of 8.32 μg/ml (Blin-1), 12.3 μg/ml (Nalm-6), 4.57 μg/ml (Daudi) and 5.01 μg/ml (NCI-H929) as measured by soft agar colony assay. Treatment with Scutellaria bacalensis at 50 μg/ml for 2 days induced apoptosis of 30% of Blin-1, 17% of Nalm-6, 20% of Daudi and 39% of NCI-H929 cells as measured by Annexin V assay. It also induced G2/M cell cycle arrest of Daudi cells. Scutellaria baicalensis also induced mitchondrial damage in 4 cell lines as measured by fluorescent emissions using the JC-1 assay. Protein expressions were examined by western blot using Blin-1 cells that were most sensitive to Scutellaria baicalensis. After treatment with 50 μg/ml of Scutellaria baicalensis for 2 days, levels of p27KIP1 increased by 152 % independently of p53, expression of the pro-apoptotic gene Bax increased by 208 %, and the anti-apoptotic proteins Bcl-2 and Bcl-XL decreased by 64 % and 38 %, respectively. Notably Scutellaria baicalensis decreased the expression of c-myc oncogene in a dose-dependent manner (80 % decrease at 50 μg/ml). Scutellaria baicalensis contains 21% of baicalin as measured by HPLC. The antiproliferative dose-response assays were repeated using baicalin and calculating the percent baicalin in the herb showed that the results nearly mirrored those of Scutellaria baicalensis, suggesting baicalin is the major anticancer component of this herb. We conclude that Scutellaria baicalensis inhibited the cell growth of B cell hematological malignancies including ALL, lymphoma, and myeloma in vitro by induction of apoptosis associated with mitchondrial damage and modulation of the Bcl family of genes. Thus, Scutellaria baicalensis and its major component, baicalin, should be tested in clinical trials for these hematological malignancies.
]Socioeconomic Factors May Select Adults Younger Than 60 Years of Age with “De Novo” Acute Myeloid Leukemia To Receive Intensive Treatment outside a Clinical Trial in Developing Countries. Experience of a Brazilian University Center.
Authors: Fagundes Evandro M. , Rocha Vanderson , Glória Ana Beatriz F. , Clementino Nelma Cristina D. , Quintão José S. , Guimaraes Joao Paulo O. , Pedroso Enio Roberto P. , Viana Marcos B.
Published: April 2020

Socioeconomic status (SES) is associated with treatment outcomes of ALL childhood. Whether this factor is associated with outcomes in adults AML in a developing country is not known. We have studied the impact of the human development index (HDI) of the United Nations as a SES factor for treatment outcomes of adults with “de novo” AML. Among 124 consecutive patients retrospectively analyzed, 46 (37 %) died during induction, 66 reached complete remission (53%) and 46 (37%) received high dose Ara-C (Hidac) consolidation. Five years-overall survival (OS) and leukemia free survival (LFS) were 17%±3% and 26%±6% respectively for all patients and 36%±7% and 30%±7% respectively for those receiving Hidac. In multivariate analysis, the HDI lower than 0.660 was associated with lower probability to receive Hidac (p=0.001), a trend for higher mortality in remission induction (p=0.062) and a decreased LFS (p<0.0001), however it was not associated with outcomes for patients receiving Hidac. In conclusion, survival for patients who received Hidac consolidation is similar to those reported in developed countries, but results of overall outcomes are inferior, probably due to the influence of socio-economic factors before Hidac consolidation. Poor SES may be a factor associated with patient selection for intensive treatment and survival.
Folate Pathway Gene Expression Differs in Genetic Subtypes of Acute Lymphoblastic Leukemia and Influences Methotrexate Pharmacodynamics.
Authors: Kager Leo , Cheok Meyling H. , Yang Wenjian , Zaza Gianluigi , Pui Ching-Hon , Downing James R. , Relling Mary V. , Evans William E.
Published: April 2020

Methotrexate (MTX) is an essential treatment component for acute lymphoblastic leukemia (ALL). The ability of leukemia cells to accumulate MTX in its polyglutamylated form (MTXPG) is recognized as an important determinant of its antileukemic effect. We measured in vivo MTXPG accumulation in leukemia cells from 101 children with ALL, and established that blasts of B-lineage ALL with either the TEL-AML1 (n=24 patients, median 911, range 338 to 5906 pmol/109 blasts) or E2A-PBX1 gene fusion (n=5, median 553, range 364 to 800 pmol/109 blasts) or T-lineage ALL (n=14, median 572, range 284 to 1468 pmol/109 blasts) accumulate significantly lower MTXPG, compared to those of other B-lineage ALL (BNHD, n=39, median 2210, range 186 to 9722 pmol/109 blasts) or hyperdiploid ALL (BHD, n=19, median 4375, range 377 to 9206 pmol/109 blasts) (E2A-PBX1 versus BHD, p=0.008; E2A-PBX1 vs. BNHD, p=0.010; TEL-AML1 vs. BHD, p<0.001; TEL-AML1 vs. BNHD, p=0.004; T-ALL vs. BHD and BNHD, p<0.001; p-values are from pair-wise comparisons using Wilcoxon rank sum test, adjusted for multiple testing using Holm’s method). To elucidate mechanisms underlying these differences in MTXPG accumulation, we used oligonucleotide microarrays (Affymetrix® HG-U133A) to analyze expression of 32 folate pathway genes (53 probe sets) in diagnostic bone marrow blasts from 197 children with ALL. This revealed ALL subtype-specific patterns of folate metabolism gene expression and identified differences in gene expression that discriminated the MTXPG accumulation phenotype in ALL cells. We found significantly lower expression of the reduced folate carrier (SLC19A1, MTX uptake transporter) in E2A-PBX1 ALL; significantly higher expression of breast cancer resistance protein (ABCG2, MTX efflux transporter) in TEL-AML1 ALL; and lower expression of FPGS (catalyzes formation of MTXPG) in T-ALL; consistent with lower MTXPG accumulation in these ALL subtypes. These findings reveal distinct mechanisms of subtype-specific differences in MTXPG accumulation and point to new strategies to overcome this potential cause of treatment failure in childhood ALL.
A Phase II Study of Temsirolimus (CCI-779) in Patients with Advanced Leukemias.
Authors: Yee Karen W.L. , Garcia-Manero Guillermo , Thomas Deborah , Ravandi-Kashani Farhad , Verstovsek Srdan , Andreeff Michael , Dancey Janet E. , Giles Francis J.
Published: April 2020

Temsirolimus (CCI-779, Wyeth Pharmaceuticals) has been shown to inhibit proliferation of a variety of tumors and induce G1 cell cycle arrest by preventing activation of the serine/threonine kinase, mTOR (mammalian target of rapamycin), a downstream effector of the PI3K/Akt pathway. Several lines of evidence implicate the PI3K/Akt/mTOR pathway in hematological malignancies. Interim results of a phase II study evaluating the efficacy and toxicity of single-agent temsirolimus in patients with advanced malignancies are presented. Temsirolimus was administered weekly, at a flat dose of 25 mg, as a 30-minute intravenous infusion. Treatment was continued until evidence of disease progression or unacceptable toxicity. To date, 8 patients have been enrolled and 7 are evaluable for efficacy and toxicity (one patient did not receive temsirolimus). Of these 7 patients, 5 had AML, 1 CML-myeloid blast crisis, and 1 ALL. Median age was 68 years (range, 21 to 87 years) and 5 were male. All patients had received prior therapy, median 2 regimens (range, 1 to 3 regimens). The median number of temsirolimus doses administered was 3 (range, 1 to 10) with a median time on study of 18 days (range, 3 to 89+ days). The most common temsirolimus-related adverse events were grade ≤ 2 and consisted of anorexia, nausea and/or vomiting and diarrhea, mucositis, dermatitis, hypertriglyceridemia, hyperglycemia, hypomagnesemia, hypocalcemia, hypokalemia, hypophosphatemia, and fatigue/asthenia. No patient developed nonspecific pneumonitis. Grade 3 toxicities included fatigue/asthenia (2), hyperglycemia (1), painful mucositis with dehydration and electrolyte abnormalities (1), diarrhea with hypokalemia (1), and hypophosphatemia (1). No patient experienced grade 4 toxicities or death from temsirolimus. No patient required dose reductions, but 2 did have dose delays due to grade 3 mucositis (1) and out-of-state hospitalization for pneumonia and atrial fibrillation (1). No patient achieved a complete remission. One patient with heavily pre-treated Philadelphia-negative precursor B-cell ALL had a transient 79% to 91% reduction in peripheral blood blasts 4 days after the first dose of temsirolimus that was maintained for 12 days prior to disease progression with involvement of the synovial fluid in bilateral knees. At the time of analysis, 6 of the 7 patients have discontinued treatment due to disease progression (4), patient refusal (1), or physician decision (1). Preliminary findings indicate that temsirolimus is relatively well-tolerated at a dose of 25 mg per week and may have biologic activity in ALL. Accrual onto the study is currently ongoing.
Down Syndrome Patients with Relapsed Acute Myelogenous Leukemia.
Authors: Loew Thomas W. , Gamis Alan , Smith Franklin O. , Massey Gita V. , Woods William G. , Hilden Joanne M. , Lange Beverly , Alonzo Todd A. , Chang Myron , Gerbing Robert B.
Published: April 2020

Down Syndrome (DS) is known to increase the risk of acute myelogenous leukemia (AML). Recent cooperative group clinical trials, POG 9421 and CCG 2891, using standard timing and cytarabine/daunomycin based chemotherapy achieved induction rates of 95% and 91%, respectively. 218 patients (57 on 9421, 161 on standard timing arm of 2891) were treated. Induction failures occurred in 17/218 (3/57 on 9421, 14/161 on 2891). Relapses occurred in 11% (24/218) [4/57 on 9421, 20/161 on 2891]. Analysis of these 24 patients demonstrate overall survival (OS) of 12% (3/24). One patient on 9421 relapsed with ALL and successfully underwent ALL chemotherapy. The remaining 23 patients had relapsed AML with OS of 8% (2/23), while 13 died of progressive disease, five of infection, two of toxicity and one of graft-versus-host disease. Median time to relapse from on-study was nine months (range 5-17). Median time to death from relapse was five months (range 1–18). Reinduction attempts were known to occur in 16 patients. Combination chemotherapy with cytarabine was the most common regimen. Seven patients achieved M1 marrows, one M2 and eight nonresponders. Mitoxantrone/cytarabine was used in 5/16 reinduction attempts with three M1 marrows, one M2 and one nonresponder. Of nine patients who underwent BMT (eight known allogeneic), two are long-term survivors (OS 22%). These patients relapsed 12 and 22 months post on-study. Both achieved second remission with induction therapy consisting of mitoxantrone/cytarabine and were consolidated with an allogeneic, related, haploidentical BMT using a busulfan/cyclophosphamide preparative regimen. Outcome for DS patients with relapsed AML is dismal. Mitoxantrone/cytarabine chemotherapy followed by allogeneic BMT may improve survival. Further evaluation of therapies is needed for this group of patients.
Incidence of Febrile Neutropenia Is Low and Unrelated to Day 1 Neutrophil Count in Hodgkin’s Lymphoma Treated with ABVD.
Authors: Chand Vikram K. , Ritchie Justine , Shannon Mary , Link Brian K. , Wooldridge James E.
Published: April 2020

BACKGROUND: When neutropenia without infection is encountered while treating Hodgkin’s Lymphoma (HL) with ABVD chemotherapy clinicians may choose to delay or reduce the chemotherapy dose or maintain the dose intensity and schedule with or without neutrophil growth factor support out of concern for neutropenic complications. We sought to determine the frequency of neutropenia and febrile neutropenia (FN) during ABVD chemotherapy.

METHODS: We examined the medical records of all patients seen at Univ. of Iowa diagnosed with HL between 1/1/1990 and 12/31/2002 treated with ABVD chemotherapy. We reviewed the charts with complete chemotherapy dosing records to determine the incidence of neutropenia, dose reduction, dose delay and chart record of febrile neutropenia (FN).

RESULTS: 218 patients were seen at UIHC with the diagnosis of HL. 104 patients were treated with ABVD chemotherapy. Adequate dosing data was available for 82 of these patients (894 treatments). On scheduled day of treatment (Day 1 and 15 of each cycle) at least Grade III neutropenia (ANC<1000) was present in 51 pts and 165 (18.45%) treatments. Grade IV (ANC<500) neutropenia was present in 28 pts and 56 (6.26%) treatments. Grade III/IV neutropenia was most frequent at treatment 2 (C1, D15) (n=32). Figure 1 illustrates the frequency of Grade III/IV neutropenia on day 1 of treatment. Dose modification (DM) defined as dose delay of > 4 days and/or dose reduction to < 80% of original doxorubicin dose following neutropenia occurred at only 10 of 165 treatments. No episodes of FN developed in this cohort. 155 treatments at the time of neutropenia were administered without dose reduction or dose delay. Growth factor support was co-administered in 55 (36.77%) of these treatments and the remaining treatments at the time of neutropenia (73.23%, n=100) were administered without growth factor support. One episode of FN developed in each of these subsets. No FN was observed following the 56 treatments administered with an ANC < 500, including 54 managed without DM (32 with GCSF support and 22 without). 672 treatments were administered with ANC ≥ 1000 resulting in 6 episodes of FN. DM was observed in 78 of these 672 treatments with 3 subsequent episodes of FN. 57 treatments (4 with DM, 23 with GCSF, 20 without GCSF, 14 unknown GCSF) had no available ANC data and were associated with one episode of FN. FN developed a total of 9 times in 8 of 82 patients over 894 treatments.

CONCLUSION: Among HL patients treated with ABVD chemotherapy we found a high frequency of neutropenia - most commonly following treatment 1. We found a very low incidence of FN that was independent of neutropenia at the time of chemotherapy administration. Dose modification for neutropenia without infection at the time of ABVD administration is not required to reduce risk of FN, and should be avoided in settings where maintaining dose intensity is considered valuable.
Biological Effects of Rabbit Anti-Thymocyte Immunoglobulin (rATG, Thymoglobulin®) in T-Cell Non-Hodgkin’s Lymphoma Cell Lines.
Authors: Hernandez Francisco J. , Reddy Nishita , Nallapareddy Sujatha , Czuczman Myron S.
Published: April 2020

Monoclonal antibodies (mAbs) have emerged as powerful adjuncts in the treatment of patients with B-cell lymphoproliferative disorders. While the treatment of B-cell lymphomas has incorporated mAbs and other biological agents into standard chemotherapy regimens, the treatment options for patients with T-cell lymphomas remain relatively limited. There exists a dire need to develop targeted therapies for T-cell lymphomas. Thymoglobulin® (rATG) is a rabbit polyclonal antibody targeting various receptors present on T-cell lymphocytes. When administered at high doses, rATG is known to deplete various subsets of T-cell lymphocytes and induce tolerance in solid organ or bone marrow transplant settings. Using several pre-clinical models, we evaluated the biological effects of rATG against various T-cell lymphoma cell lines. Experiments were conducted in HH, H9, Loucy and HT102 cell lines. A B-cell mantle cell lymphoma cell line was used as a control (MJ). rATG-induced cell-growth inhibition was measured by [3H]-Thymide incorporation assays and measured at 24 and 48 hours. Induction of apoptosis in T-cell lines following rATG exposure was determined by annexin-V/propidium iodine staining and quantified by flow cytometric analysis. Standard functional assays for ADCC/CMC were performed using rATG (5 or 25mg/ml) in 51Cr-labeled T-cells. We found that rATG inhibited DNA synthesis in all the T-cell lines tested. No biological effect was observed in the B-cell mantle cell lymphoma line. Treatment with rATG at either 5 or 25mg/ml resulted in a 30 to 50% growth inhibition when compared to isotype or vehicle controls (P<0.05). Induction of apoptosis was demonstrated in 30 to 40% of T-cell lymphoma cells 24 hrs following exposure to ATG. Biological effects of rATG were dose-dependent. In addition, rATG induced significant ADCC and CMC in T-cell lymphoma cell lines. In conclusion, our data demonstrate that rATG is active against a variety of T-cell lymphoma cell lines in vitro. Anti-tumor effects of rATG are mediated by induction of direct signaling and via the activation of the innate immune system. Additional in vivo studies using T-cell lymphoma are underway and will be presented at the annual meeting.
Preferential Induction of B Cell Apoptosis Using Photodynamic Therapy.
Authors: Krosl Gorazd , Dube Pascale , Dallaire Nancy , Vaillancourt Marc , Roy Denis-Claude
Published: April 2020

Apoptotic cell death in cancer cells promotes antigen presentation and favors the development of an anti-tumor vaccination effect. While lymphoma cells can easily be obtained from lymph nodes or peripheral blood, these malignant cells usually co-localize with normal hematopoietic T lymphocytes, rendering the selective induction of apoptosis in malignant B cells particularly difficult. We have found that photodynamic therapy (PDT) using the photosensitizer 4–5 dibromorhodamine methyl ester (TH9402) and visible light (514 nm) can be used to preferentially eliminate malignant B-lineage non-Hodgkin’s lymphoma cells (mean of 3–4 logs) over T lymphocytes. As a rhodamine derivative, TH9402 cellular efflux is mediated through the P-glycoprotein (Pgp) pump. However, the preferential elimination of B cells over resting T cells could not be ascribed to Pgp inactivation and we found no difference in intracellular retention of TH9402 between B and T cells. In order to understand the molecular events leading to such high B cell susceptibility to PDT, we then investigated the nature of death pathways involved. To determine whether apoptosis was the preponderant mechanism leading to the rapid elimination of B cells, we measured the binding of Annexin V (AnnV) in 7-aminoactinomycin D (7AAD) negative (i.e. live) B and T cells using flow cytometry, and quantified DNA fragmentation and caspase 3 and 9 activity in isolated CD3+ and CD19+ PDT treated and untreated cells. We found that B cells demonstrated high levels of apoptosis after PDT, with 53±1.3% (mean±SE) AnnexinV+/7AAD- cells compared to 30±1.9% in T cells (p< 0.05). The extent of DNA fragmentation, as determined by the TUNEL reaction, was extensive in B cells (33.7±9.8%)(mean±SE) but not T cells (2.3±0.1%) when measured at 4 hours post PDT (p<0.01). After PDT, catalytic caspase 3 levels were also 4-fold higher in CD19+ cells than CD3+ cells (p<0.01). In contrast, caspase 9 levels remained low for both cell types. The induction of apoptosis translated into the elimination of more than 90% of B cells within 4 hours after PDT, while the majority of T cells were not affected. Importantly, similar elimination of B-lineage cell lines and patient cells was observed, using a limiting dilution assay, whether cells overexpressed the anti-apoptotic bcl-2 protein or not. Thus, these results indicate that the predominant caspase 3 apoptotic pathway is specifically implicated in PDT-induced B cell death, and its early activation explains the rapid and profound sensitivity of B lymphocytes to PDT. This strategy could promote tumor cell vaccination in the context of autologous TH9402-purged stem cell transplantation for patients with B-lineage malignancies. In addition, preferential B cell targeting could take advantage of the crucial immunomodulatory role of apoptosis for the treatment of patients with autoimmune B cell disorders.
Phase I Clinical Trial of Glivec in Combination with Tetra-Arsenic Tetra-Sulfide in the Treatment of CML Patients in Advanced Phase.
Authors: Li Jun-Min , Wang Ai-Hua , Sun Hui-Ping , Shen Yang , Zhao Rui-Hua , Gu Bai-Wei , Chen Bing , Xing Wen , Shen Zhi-Xiang , Wang Zhen-Yi , Chen Sai-Juan , Chen Zhu
Published: April 2020

OBJECTIVE: To evaluate the safety and efficacy of Glivec in combination with As4S4 in the treatment of advanced CML patients.

PATIENTS AND METHODS: 9 Ph+ CML patients in advanced phase were entered into the study. The first 3 patients received Glivec 400mg/d plus As4S4 150mg/kg/d (day 1–14, each month). If no patient were to experience grade 3/4 CTC toxicity after one month of treatment, the dose of Glivec would be increased to 600mg. All the patients were treated and followed up for 6 months. Adverse events, hematological, cytogenetic, molecular response and real-time RT-PCR were evaluated in each regular visit.

RESULTS: From Dec.2003 to June.2004, 9 patients consisting of 6 males and 3 females were enrolled in this trial, 5 in accelerated phase and 4 in blast crisis, aged 22–53 years (median 33). The first 3 patients received Glivec 400mg/d plus As4S4, and other 6 patients received increased dosage of Glivec to 600mg/d. By the time of report, 6 patients had finished the study. 7/9 patients (77.8%) achieved complete hematological response (CHR). 2 patients relapsed after 4 and 5 months of treatment, respectively, and 2 patients withdrew from the study after 1 and 2 months respectively to receive chemotherapy due to progression of the disease. Notably, 3 of these 4 patients had complex cytogenetic abnormalities. Cytogenetic response was observed in 3 patients (33.3%), including 2 major cytogenetic response (1 complete response) and 1 minor response. No molecular response was obtained so far in any patients. Bcr-Abl fusion transcript copy number was measured by real-time RT-PCR in 3 patients, 1 patient with CCR had 3140 fold reduction of BCR-ABL DoseN. Non-hematologic toxicities such as gastrointerstinal discomfort, edema, liver dysfunction and myalgia/bone pain were common and mild (grade 1/2). Grade 3/4 hematological toxicities including neutropenia, thrombocytopenia and anemia occurred in 3 patients.

CONCLUSIONS: Glivec in combination with As4S4 has potential effect in advanced CML with acceptable toxicities. Further investigation will be performed to compare the efficacy of Glivec/As4S4 combinative therapy with Glivec mono-therapy in advanced CML.
Immunogenecity of CML-DC Is Maintained upon Imatinib Mesylate: Implication for Vaccination Regimens.
Authors: Westers Theresia M. , Janssen Jeroen J.W.M. , Snoijs Niels C.L. , van de Loosdrecht Arjan A. , Ossenkoppele Gert J.
Published: April 2020

Chronic myeloid leukemia (CML) is characterized by the presence of the Philidelphia chromosome which encodes for the fusion protein bcr-abl, a constitutively active tyrosine kinase. Imatinib mesylate (Gleevec, Novartis) is an inhibitor of the kinase activity of bcr-abl and therefore a treatment modality for CML. High rates of cytogenetic responses are found, although in many patients minimal residual disease (MRD) is detected by molecular techniques. Immunotherapy using leukemic dendritic cell (DC) based vaccination might be a feasible approach to eradicate MRD. In a pilot study on CML-DC-based vaccination in advanced CML DTH responses towards CML cells were achieved. (Ossenkoppele et al., Leukemia, 2003, 17, 1424). However, little is known about the effects of Imatinib mesylate on bcr-abl positive CML-DC during vaccination. In this study we investigated effects of Imatinib on culture of CML-DC, their viability, T cell stimulating and migratory capacity in vitro in 17 patients. Imatinib hardly affected the immunophenotypical profile of CML-DC. Expression of CD40, CD80, CD83, HLA-DR and chemokine receptors (CCR5, CCR7, CXCR4) remained stable during overnight exposure. Only the fluorescence intensity of CD86 was significantly higher (1–10μM, p=0.043) and that of CD54 significantly decreased in the highest Imatinib concentration tested (p=0.043). Exposure of Imatinib-free cultured CML-DC to Imatinib significantly reduced the recovery of viable cells and hence the DC yield in a dose-dependent manner (5.3–26% reduction after 20h. for 1–10μM, p=0.043). Migration of mature CML-DC towards CCL19 (MIP3β) was significantly improved in the presence of 1 and 5μM Imatinib which implies increased ability to reach the lymph nodes (p=0.043, mean 27, 36 and 34% migration for 0, 1 and 5μM Imatinib, respectively). Imatinib did not affect T cell stimulating capacity of CML-DC, except at concentrations higher than 3μM (p=0.028). No significant changes were observed for Imatinib exposure of CD34+ DC isolated from normal subjects. In conclusion, Imatinib at low concentrations maintains the immunogenecity of CML-DC. Since Imatinib plasma levels in CML patients are 1.5 and 3.0μM upon 400 and 800mg daily, respectively, our data justify continuation of Imatinib treatment during CML-DC-based vaccination regimens.
Disease Models and Transformation Mechanisms Mediated by MLL-AF4 Family Oncoproteins in Human Leukemia.
Authors: So Chi Wai Eric , Wong Piu , Lin Min , Cleary Michael L.
Published: April 2020

The Mixed Lineage Leukemia (MLL) gene codes for a histone methyltransferase that is required for hematopoietic development. As a consequence of chromosomal translocations, MLL is fused with over 40 different genes to yield in-frame fusion proteins in acute leukemias. AF4, the most common fusion partner, accounts for 40% of MLL leukemias. The AF4-related proteins, LAF4 and AF5q31, are also fusion partners for MLL in rare cases of leukemia, whereas a fourth family member, FMR2, is a candidate protein for fragile X mental retardation syndrome. MLL fusions with AF4 family proteins manifest as acute biphenotypic or lymphoblastic leukemias that are associated with a poor prognosis, but the lack of appropriate disease models has hampered progress in understanding their underlying molecular mechanisms. Here we report that each of the MLL-AF4 family fusion proteins (the leukemia associated MLL-AF4, MLL-LAF4, and MLL-AF5q31 as well as an artificial MLL-FMR2) is capable of altering the growth and self-renewal properties of primary murine hematopoietic stem/progenitor cells in a serial methylcellulose culture myeloid replating assay. However, significant differences were observed in their respective oncogenic potentials. Cells transduced with MLL-LAF4, MLL-AF5q31 and MLL-FMR2 rapidly expanded during serial replatings, adapted to growth in liquid culture, and induced leukemias in syngeneic recipient mice within 6 months. Conversely, bone marrow cells transduced with MLL-AF4 yielded modest numbers of blast-like colonies in the third round of plating, only transiently expanded in vitro, and quickly underwent terminal differentiation. MLL-LAF4 and MLL-AF5q31 were also capable of enhancing the self-renewal of transduced cells with B lymphoid progenitor phenotypes. A structure/function analysis demonstrated that the previously reported transcriptional transactivation domains of AF4 family proteins were neither necessary nor sufficient for in vitro transformation. Furthermore, the recently reported AF9 interaction motif that is conserved in all AF4 family proteins was also shown to be dispensable for in vitro transformation. These data indicate that transactivation per se, and interaction with AF9 in particular, are not absolutely required for the oncogenic actions of MLL-AF4 family proteins. Conversely, a minimal transformation domain was mapped to the highly conserved carboxy-terminal homology domain shared among all AF4 family proteins, including the Drosophila homolog Lilliputian, and shown to be necessary and sufficient for transformation. Taken together, our studies establish transformation models for MLL-AF4 family fusion proteins and provide critical mechanistic insights into their underlying molecular mechanisms.
Generation of Resistance Cell Lines to AMN107, a New Inhibitor of BCR-ABL and Its Effects on Cell Lines Sensitive and Resistant to Imatinib.
Authors: Mahon Francois-xavier , Lagarde Valerie , Manley Paul W. , Pasquet Jean-Max , Turcq Beatrice , Reiffers Josy
Published: April 2020

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myelogenous Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Imatinib is a selective inhibitor of Bcr-Abl tyrosine kinase and is now used in frontline therapy for CML. However clinical resistance is the main concern using this treatment, mediated by mutations within the kinase domain of Bcr-Abl, amplification of the BCR-ABL genomic locus or other as yet unknown mechanisms. AMN107 (Novartis Pharma AG, Basel, Switzerland) is a synthetic, second generation inhibitor of Bcr-Abl tyrosine kinase. In the current study, we tested AMN107 against different Bcr-Abl positive cell lines such as K562, LAMA84, AR230 or murine Ba/F3 cells transfected with BCR-ABL (BaF/BCR-ABL). In a 4 day proliferation assay (MTS) the dose of AMN107 that inhibited 90% of the cells (IC90) was 0.01 μM which was 100-fold lower than the IC90 of imatinib. In addition, proliferation of imatinib resistant cell lines which exhibited amplification of BCR-ABL was inhibited by 75% in the presence of 0.01 μM AMN107. However, Ba/F3 cells expressing the imatinib resistant BCR-ABL T315I mutant were only inhibited with 10μM of AMN107. Furthermore, K562-R, an imatinib-resistant cell line exhibiting a new mechanism of imatinib resistance (modification of chaperone proteins such as heat shock proteins) was also insensitive to AMN107; the IC90 for AMN107 at day 4 was 1μM versus 4μM for imatinib.

Finally, we investigated potential resistance to AMN107 in Bcr-Abl positive cells. Resistant cell lines were generated after long-term (2 month) gradual dose-escalation exposure to the inhibitor. Up to now, we have obtained four cell lines, AR230-ra, K562-ra, LAMA 84-ra, and BaF/BCR-ABL-ra resistant to 8, 10, 10, and 100 nM of AMN107, respectively. Resistance was defined as the capacity to survive in the continuous presence of doses of AMN107 that kill in three days more than 90% of the parental cells in liquid culture.

Preliminary investigations of AMN107 resistance using western blot and cytometry have shown that only BaF/BCR-ABL-ra overexpressed wildtype Bcr-Abl as we have already reported for imatinib resistance. Studies with these resistant cell lines investigating cross resistance with imatinib and looking for additional mechanisms of resistance are in progress.We conclude that in vitro, AMN107 is more powerful than imatinib in inhibiting the proliferation of BCR-ABL positive cell lines. In addition, we have demonstrated that it is possible to develop resistant cell lines to this new inhibitor of Bcr-Abl.
Effect of the Combination of Imatinib Mesylate (Glivec) and Curcumin in Chronicm Myeloid Leukemia Cell Line.
Authors: Kim Young-Ju , Choi Yong-Hee , Kim Chang Up , Bae Eun Kyung , Kim Byung-Su , Ahn Kwang-Sung , Lee Jong-Seok , Park Seonyang , Kim Byoung Kook , Yoon Sung-Soo
Published: April 2020

Imatinib mesylate (Glivec, Gleevec, STI571) is highly effective in patients with chronic myeloid leukemia (CML). Despite high rates of hematologic and cytogenetic responses to Glivec therapy, the emergence of resistance to Glivec has been recognized as a major problem in the treatment of CML. Those problems leaded us to search for novel therapy, combining curcumin with Glivec. Recently we reported that curcumin induced apoptosis via repression of p-AKT and activation of caspase-3. To investigate whether combination of curcumin with Glivec is more effective than Glivec alone, the expression of apoptosis related genes, such as Bcl-2 family, caspase-3, and its cell cycle proteins, such as p53, p21, cyclin D, cyclin A, cyclin B1 and PCNA were examined by western analysis in K562 chronic myeloid leukemia cell line. Our results showed that Bcl-2, -XL, and Bid were more down-regulated with combined treatment compared with single treatment controls (either Glivec or curcumin alone). Also, the activity of caspase 3 was 2-fold higher than compared with control groups. In western analysis, procaspase 3 was dramatically reduced with combined treatment. Also, cell cycle proteins except p53 were more down-regulated in combined treatment. For further investigation of the genes affected by combination therapy, DNA chip analysis was performed. The expression of candidate genes, which we selected in chip analysis, was confirmed using real-time PCR. Our result indicated that apoptosis related genes were up-regulated and cyclin D1 and cyclin A were down-regulated. Also, several genes (protein phosphatase 2C, ZNF272, protein-tyrosine phosphatase nonreceptor 7 (PTPN7), SOCS2) were more down-regulated in combined treatment, showing 2-fold difference. Those genes may be associated with the progression of chronic myeloid leukemia. We suggest that their down-regulation by combined treatment was effective mainly via the induction of apoptosis. Taken together, our data suggested that combined treatment with curcumin could be effective to overcome the problems of Imatinib mesylate (Glivec, STI571) therapy.