bluuebunny/crossref_metadata_2025 huggingface dataset:
Imatinib Mesylate for Patients (pts) with Hypereosinophilic Syndrome (HES) and Systemic Mastocytosis (SM): A Phase II Trial.
Authors: Jabbour Elias , Kantarjian Hagop , Verstovsek Srdan , Jones Dan , O’Brien Susan , Luthra Raja , Ravandi-Kashani Farhad , Wierda William , Giles Francis , Rios Mary Beth , Cortes Jorge
Published: April 2020

Imatinib is a potent inhibitor of the tyrosine kinases c-abl, c-kit, ARG, platelet-derived growth factor (PDGFR)-alpha and PDGFR-beta. We and others have reported on the clinical efficacy of imatinib in pts with HES and SM. We report here on 28 pts (16 HES, 12 SM) treated in a phase II study of imatinib. Pts with HES received imatinib at a starting dose of 100 mg daily with escalation to 400 mg if no response after 1 month (mo). Pts with SM were started at a dose of 400 mg with possible escalation to 800 mg. The median age was 52 years (yrs) (26–77 yrs). All pts had signs and symptoms of end-organ involvement with 10 (83%) of 12 pts with SM having skin involvement. 15 (53%) of 28 pts had received prior therapies. Imatinib was started within 34 mo from diagnosis (3–162 mo). Prior to the start of imatinib, the median eosinophil count for pts with HES was 2.2 x109/L (0.5–7 x109/L) and the median percentage of eosinophils in the bone marrow was 19% (5%–28%). 2 of the pts with SM had increased eosinophils, 9 pts have been evaluated for serum tryptase level; this was increased in 5 (0 HES, 5 SM). 6/16 (37%) pts with HES responded: 3 achieved a complete remission (CR) (defined by the disappearance of eosinophilia and signs and symptoms of disease) and 3 a partial response (PR) (defined by the reduction of eosinophilia and organomegaly by > 50%). Two pts (1 CR, 1 PR) required dose escalation to 400 mg. The median duration of response was 11+ mo (1+ to 21+). One patient died in CR at 18 mo, 1 was lost to follow-up after 1 mo in CR; 2 have ongoing response (1 PR, 1 CR) 15+ and 21+ mo, respectively; and 2 lost PR after 3 and 10 mo, respectively despite dose escalation. 7/12 pts with SM (58%) responded. Six pts achieved a major response (MR) (defined by the complete resolution of at least one clinical finding and no progression in other clinical findings): 4 CRs, 1 PR (defined by the incomplete regression of one or more clinical finding(s) without complete regression and without progress in other clinical findings) (Leuk Res 27:635,2003). One pt (MR) required imatinib escalation to 800 mg. The median duration of response was 14+ mo (7+ to 36+). At the time of response, 2 pts had normalized their tryptase levels. Only one pt has lost response (PR) after 9 mo. Overall, treatment was well tolerated. 2 pts experienced grade 3 diarrhea; one of them required imatinib dose reduction. We conclude that therapy with imatinib for patients with HES and SM appears effective and well tolerated.
Histidine Decarboxylase (HDC) as Novel Marker of Immature Neoplastic Mast Cells in Systemic Mastocytosis.
Authors: Krauth Maria T. , Agis Hermine , Aichberger Karl J. , Simonitsch Ingrid , Muellauer Leonhard , Mayerhofer Matthias , Horny Hans P. , Valent Peter
Published: April 2020

Synthesis of histamine in hematopoietic progenitor cells may be one of the earliest events in mast cell-development from pluripotent hematopoietic progenitor cells. In the present study, we asked whether the key enzyme involved in histamine generation, histidine decarboxylase (HDC), can be employed as an immunohistochemical marker for the detection of (neoplastic) mast cells (MC) in patients with cutaneous (CM) or systemic mastocytosis (SM). To address this question, we examined bone marrow biopsy specimens in a cohort of 101 patients (CM, n=10; indolent SM, n=46; SM with associated clonal non-MC lineage disease, n=31; aggressive SM, n=10; MC leukemia, n=3; MC sarcoma, n=1) using an antibody against HDC. Independent of the maturation stage of MC or subtype of disease, the anti-HDC antibody produced clear diagnostic staining results in all patients with bone marrow involvement examined including those with MC leukemia and MC sarcoma, in which MC are particularly immature and often escape analysis when examined by conventional stains. In these patients (MC leukemia, n=2; MC sarcoma, n=1), expression of HDC was reconfirmed at the mRNA level by RT-PCR analysis performed with RNA of highly enriched sorted CD117+ MC. In patients with CM or normal/reactive bone marrow (n=30), no HDC-positive infiltrates were detected. In these patients, only a few hematopoietic cells, presumably basophils, were found to react with the anti-HDC antibody. In summary, HDC is expressed in neoplastic bone marrow MC in patients with SM independent of the maturation stage of cells or the variant of disease. HDC should therefore be considered as a new MC marker in the screen panel of antigens employed to diagnose high grade MC malignancies.
WT-1 Over-Expression Could Be Related to " High Metaplastic Index" in Myelofibrosis with Myeloid Metaplasia (MMM) Patients. A Preliminary Report.
Authors: Iuliano Francesco , Cilloni Daniela , Gottardi Enrico , Fava Milena , Fabiano Flora , Peta Antonio , Saglio Giuseppe
Published: April 2020

WT1 is a tumor-suppressor gene coding for a zinc-finger transcription factor located on chromosome 11p13, which was originally identified for its involvement in the pathogenesis of the Wilms’ tumor. In normal bone marrow and peripheral blood(PB)WT1 expression is extremely low and often undetectable even by RT-PCR. By contrast WT1 expression has been previously reported to be increased in MDS, AML, ALL and PNH. In CMPDs, little data are available only in CML. With this background we analyze by RQ-PCR the WT1 transcript amount in 13 patients with myelofibrosis with myeloid metaplasia (MMM) in comparison with a group of 32 age- and sex-matched healthy controls. All measurements were carried out only on PB and performed as previous published. All MMM patients were under cytoreductive treatment and at different time from diagnosis ( range 1 – 7 yrs). In MMM WT1 transcript amount is consistently higher (range 23–2518 WT1 copies/104 ABL copies, mean 382±692) than controls ( range 0 – 22, mean 5,6 ± 5,87) ( P < 0.0001 Mann-Whitney U test). To assess the significance of the WT1 expression as a marker for disease features, the following variables were analyzed and compared : age, hemoglobin level, white blood cell count, circulating blasts, LDH serum levels, number of peripheral CD 34 cells, Dupriez score and, according to Barosi published data, myeloproliferation index, myelodepletion index, severity score. We did not found any significant correlation between WT1 and all these variables, probably because the patients were studied during the course of their disease. Moreover, cytoreductive treatment might have modified the disease characteristics. Stricking, when analyzed WT1 expression according to the therapeutic need, we found a subset of patients with a WT1 overexpression ( range 132–2518, mean 758±911; p <0.06) and spleen size between 4 – 7 cm despite receiving > 1,5 gr/daily of hydroxyurea or more than 1 drug. In such of patients WT1 over-expression allowed an almost complete discrimination between MMM in stable phase and MMM with “high metaplastic index”. In conclusion, our results, although based on a relatively small number of patients, provide an interesting profile of WT1 expression in MMM. The possible clinico-therapeutic implications of these observations are worthy of investigation in well-designed studies.
A Phase II Trial of Imatinib Mesylate (Gleevec®) in Myeloproliferative Disorders Other Than CML.
Authors: Kuriakose Philip , Shurafa Muhammad S.
Published: April 2020

Myeloproliferative disorders are a heterogeneous group of disorders characterized by increased cell numbers and variable degrees of organomegaly and myelofibrosis. To date, the standard of care has been phlebotomy followed by Hydroxyurea or anagrelide to control thrombocytosis. Anagrelide has gained popularity in recent years due to its theoretical non leukemogenic potential. Gleevec® is a small molecule inhibitor of several tyrosine kinases including c-kit and platelet derived growth factor receptor (PDGFR). The latter is expressed on platelets and megakaryocytes and may offer an explanation for the megakaryocytic hyperplasia and clustering via an autocrine loop action. In vitro, Gleevec® inhibits spontaneous burst formation in polycythemia vera (PV) probably by inhibiting c-kit. By inhibiting PDGFR, it could inhibit megakaryocytic proliferation by blocking an autocrine effect which may be responsible for megakaryocytic hyperplasia and clustering in essential thrombocytosis (ET) and PV. In 2003 we initiated a study to explore the effectiveness of Gleevec® in these two entities. 11 patients have been enrolled, 5 with PV, and 6 with ET. The starting dose in each case was 400mg, with escalation to 800 mg based on response. Complete response (CR) was defined as therapeutic range hematocrit without phlebotomy, a normal platelet count and discontinuation of Hydrea or Anagrelide. There were two CR’s and one PR in PV. All three patients discontinued treatment because of toxicity at 2, 6, and 10 months, respectively. There was one CR and one PR in ET, but both discontinued treatment due to toxicity at 7 months and non-compliance at 2 months, respectively. The toxicities observed that led to discontinuation of therapy were grade 2–3 diarrhea (3/11), grade 2–3 periorbital and ankle edema (5/11), grade 2 skin rash (1/11), grade 1 blurred vision (1/11). One major post operative bleeding was observed and was probably related to platelet dysfunction. Two patients (one each of PV & ET) had to discontinue therapy for lack of response at maximum tolerated dose. The ending dose was 400 mg (3), 600 mg (5), and 800 mg (3), with two at the 800 mg dose level being non-responders, and all others having discontinued therapy on account of toxicity. This study suggests that Gleevec® has activity in PV and ET, but toxicity at the studied dose escalation schedule could lead to discontinuation of therapy. However, this does not preclude its use in a new clinical trial with a more tolerable dosing schedule over a longer period of time.
Regulation of Berg-36 Gene Expression by Survival and Apoptotic Stimuli in B-Chronic Lymphocytic Leukaemia Cells.
Authors: Jewell Andrew P. , Baou Maria , Yong Kwee L. , Carr Robert , Murphy John
Published: April 2020

Chronic lymphocytic leukemia is characterized by the accumulation of monoclonal malignant CD5+ B cells that show resistance to apoptosis in vivo. Berg 36 (ZFP36L1, TIS11b, BRF1, cMG1, ERF1) is a zinc finger containing early response gene that was cloned from PMA-stimulated B chronic lymphocytic leukemia cells. Induced expression of this gene has been linked to calcium ionophore and anti-CD20-induced apoptosis of human B lymphoma cells. Berg36 protein has, in common with two other members of the small gene family (TIS11, TIS11d), been reported to function as an mRNA-binding protein that may promote instability of cytokine mRNAs. We have therefore studied the regulation of Berg36 expression in B-CLL cells in vitro, in response to cytokines and other signals that regulate apoptosis. B-CLL cells from 12 patients were purified and incubated with the following agents either alone or in combinations for a number of hours; IL-4, CD40 ligand, PMA or anti-CD20 antibody (rituximab). Apoptosis was measured after 24 hours by Annexin/PI staining and Berg-36 expression by Northern blot analysis. Spontaneous apoptosis in unstimulated B-CLL cells was 20.9±5.1%. Co-incubation of B-CLL cells with IL-4 reduced the percentage of apoptotic cells to 6.2±1.2%, but had no effect on Berg-36 expression. In contrast, both PMA and CD40 stimulation reduced the percentage of apoptotic cells (to 14.8±3.4% and 10.2±4.2% respectively), and markedly induced Berg36 expression. On the other hand, anti-CD20 antibody induced apoptosis (36.2±6.6%), but also induced Berg36 expression. Specific inhibitors of various intracellular signalling molecules confirmed that induction of Berg36 by different agents was mediated through different signalling pathways. In conclusion, expression of Berg36 can be induced in B-CLL cells by stimuli that induce either survival or apoptosis in these cells.
Blood Flow Cytometry Allows Us To Dispense with Bone Marrow Biopsy in the Evaluation of Treatment Response in CLL.
Authors: Maloum Karim , Charlotte Frederic , Divine Marine , Cazin Bruno , Lepretre Stephane , Delmer Alain , Sonneville Sylvie , Merle-Beral Helene
Published: April 2020

Aims: To evaluate the diagnostic value of bone marrow biopsy (BMB) with immunohistochemistry studies compared to blood flow cytometry (FC) in response evaluation after treatment in CLL. Patients and methods: 75 previously untreated patients with stage B and C-CLL were treated with oral Fludarabine (30mg/m2/d) + Cyclophosphamide (200mg/m2/d) combination, day 1 through 5, repeated every 28 days for 6 courses. Response evaluation was performed 2 months after the completion of therapy, according to NCI criteria. Sixty patients out of 75 achieved partial or complete remission and underwent BMB. Minimal residual disease (MRD) was simultaneously evaluated on paraffin-embedded BMB by anti-CD5, -CD20 and -CD3 immunostaining and in the peripheral blood using a very high sensitive FC method (Maloum et al, Br J Haematol, 2002, 119:970–5). BMB immunostaining was considered positive for MRD in case of concurrent expression of CD20 and CD5 by the same cells assessed on consecutive sections. Twenty nine patients could have been analyzed so far. The median follow-up was of 49 months [range 19–56].

Results: In all these 29 patients, BMB was considered histologically normal according to the NCI criteria. In 23/29 cases, bone marrow immunostaining did not show expressing CD20 cells. Fifteen out of 23 were in phenotypic remission using blood FC (PhR), of whom only 2 relapsed (at 31 and 37 months). The remaining 8/23 patients did not achieve PhR and all relapsed (at 22, 27, 37, 40, 45 and 51 months). In 6/29 patients, immunochemistry on BMB showed persistent B CD5+ cells : 3 did not achieve PhR of whom 2 relapsed (at 32 and 37 months) while 3 were in complete PhR and displayed a persistent clinical complete remission with a follow-up of 39, 47 and 47 months respectively. Interestingly, in the latter 3 cases, FC analysis showed an expansion of polyclonal BCD5+ cells in blood as assessed, which could explain the presence of such normal BCD5+ cells in the bone marrow. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy (ACC) to predict relapse for BMB and FC are given in the following table:

Sensitivity (%) Specificity (%) PPV (%) NPV (%) ACC (%) BMB 16.7 76.5 33.3 56.3 51.7 FC 83.3 94.1 90.9 88.9 89.7

Conclusion: These results strongly suggest that BMB should not be indicated in the evaluation of treatment response and is not useful for relapse prediction. In contrast, blood flow cytometry constitutes the best method in routine laboratory to predict clinical outcome and should be included in response treatment criteria in CLL.
Is Zap-70 Protein Detection by Flow Cytometry a Reliable Tool in CLL Prognostic Evaluation?.
Authors: Le Garff-Tavernier Magali , Ticchioni Michel , Letestu Rémi , Brissard Martine , Davi Frédéric , Sémichon Monique , Vuillier Françoise , Cymbalista Florence , Merle-Béral Hélène
Published: April 2020

Background : Expression of ZAP-70 protein has been shown to be correlated with mutational status of immunoglobulin heavy-chain variable region (IgVH) genes, a major prognostic factor in CLL. We investigated whether the detection of ZAP-70 protein by flow cytometric analysis using unconjugated and conjugated monoclonal antibodies (mAbs) could be applied securely in the workup of patients with CLL. Methods: Flow cytometric analysis of ZAP-70 protein was performed using the method described by Crespo et al (N Engl J Med2003;348:1764) with minor modifications. Both fresh and cryopreserved mononuclear cells from CLL patients and healthy donors were fixed and permeabilized using Fix and Perm kit (Caltag Laboratories), incubated with anti-ZAP-70 mAb (clone 2F3.2, Upstate Biotechnology) and then revealed with goat antimouse FITC mAb (Immunotech). Finally cells were incubated with CD3-APC, CD56-APC and CD19-PC5. We also tested 3 mAbs conjugated to various fluorochromes: 2F3.2-FITC (Upstate), 1E7.2-PE (eBioscience), 1E7.2-PE or -Alexa 488 (Caltag). ZAP-70 protein detection in B-cells was expressed either as a percentage of its expression in the T and NK-cells or as a ratio (R) of T-cell mean cell fluorescence (MCF) to B-cell MCF. Western blotting of protein lysates from purified B-cells was carried out to control results obtained by cytometry in 55 samples. Mutational status was defined using a cutoff of 98%. Results: In 13 healthy donors, the mean percentage of ZAP-70 protein expression obtained by flow cytometry with unconjugated mAb (clone 2F3.2) was 4.69% ± 1.93 [range 2–9%] and the R ratio was 6.64 ± 1.54 and > 4.8. In 83 B-CLL samples, ZAP-70 expression was determined using the same method and compared to IgVH mutational status. Results in table below show a 75% concordance between gene mutations and ZAP-70 expression when considering a percentage of positive B-cells > 20%. A better concordance (81%) is obtained with a threshold T-cell MCF/ B-cell MCF at 4 determined by Youden’s index. To note the high concordance (90%) between unmutated status and ZAP-70 + expression (19/21). Comparison with at least 1 of the 3 conjugated mAbs has been performed for 63 samples, with discordant results in our laboratories.

62 mutated IgVH samples 21 unmutated IgVH samples ZAP-70 B-Cells + ≤ 20 % : 43 > 20% : 19 T-cell MCF/B-cell MCF ≥ 4 : 48 < 4 : 19

Conclusions: Our data document the concordance between IgVH gene mutational status and ZAP-70 protein expression measured by flow cytometry, particularly in ZAP-70 negative samples. We found that the indirect method of labelling with unconjugated anti-ZAP-70 mAb remains until now, in our hands, the gold standard method compared to the available dyes conjugate mAbs.
Flow Cytometric Characterization of Genetically Defined Subgroups of Chronic Lymphocytic Leukemia: Basis for the Comprehensive Definition of Prognostic Parameters.
Authors: Kern Wolfgang , Voskova Daniela , Schoch Claudia , Hiddemann Wolfgang , Schnittger Susanne , Haferlach Torsten
Published: April 2020

Chronic lymphocytic leukemia (CLL) is defined based on cytomorphology and immunophenotype. The most important prognostic parameters are cytogenetic aberrations and the expression of CD38 and ZAP70. We analyzed correlations between immunophenotypic patterns and genetic aberrations in 153 peripheral blood samples from patients with newly diagnosed and untreated CLL. Immunophenotyping was performed applying five-fold staining, a comprehensive panel of antibodies, and CD45-SSC-gating. Genetic aberrations were detected by fluorescence in-situ hybridization using probes targeting ATM for the detection of 11q-, D12Z3 for trisomy 12, D13S319 and D13S25 for 13q-, and p53 for 17p-. As compared to cases without trisomy 12, in cases with trisomy 12 the expression levels were higher for CD22 (66% vs. 34%, p=0.001), CD20 (83% vs. 71%, p=0.034), and FMC7 (28% vs. 7%, p=0.000001). This is in accordance with our finding that higher numbers of prolymphocytes have been found in CLL with trisomy 12. As compared to cases with any cytogenetic aberration, cases with normal cytogenetics had lower expression levels of CD5 (82% vs. 89%, p=0.012) and CD19 (79% vs. 85%, p=0.051). Importantly, there were no significant differences in expression levels of CD38 and ZAP70 between cytogenetically defined subgroups of CLL. Thus, in cases with 11q-, trisomy 12, 13q-, homocygous 13q-, 17p-, and normal cytogenetics CD38 was expressed in 51%, 47%, 37%, 33%, 24%, and 38% and ZAP70 together with cyCD79a was expressed in 17%, 11%, 13%, 13%, 22%, and 15%. These data indicates that, with the exception of trisomy 12, the cytogenetic findings are not reflected by specific immunophenotypic features. In particular, this is true for the prognostically and clinically relevant markers CD38 and ZAP70 the expression of which cannot be deduced from cytogenetics. In a second step, correlations between the expression of CD38 and ZAP70 and other immunophenotypic markers have been analyzed. Cases positive for CD38 (more than 30% positive cells) had lower expression levels of CD19 (78% vs. 87%, p=0.005), CD20 (65% vs. 75%, p=0.023), and CD23 (62% vs. 74%, p=0.005). Cases positive for ZAP70 (more than 20% positive cells with coexpression of cyCD79a) had higher expression levels of CD19 (87% vs. 79%, p=0.011), CD22 (40% vs. 26%, p=0.020), and cyCD79a (75% vs. 59%, p=0.009). These findings are in line with both CD38 and ZAP70 being markers that reflect different biologic backgrounds of CLL which also reveal prognostic information. Taken together, these data strongly suggest that studies evaluating different treatment approaches in CLL should provide a detailed characterization of the analyzed patients including both cytogenetic aberrations and immunophenotypic findings as well as novel genetic markers in order to guarantee the detection of subgroup-specific treatment effects.
The Natural Product Honokiol Induces Caspase-Dependent Apoptosis in Chronic Lymphocytic Leukmemia (CLL) Cells.
Authors: Battle Traci E. , Arbiser Jack , Frank David A.
Published: April 2020

CLL remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Honokiol is a natural product known to possess potent anti-neoplastic and anti-angiogenic properties. We examined whether Honokiol can overcome apoptotic resistance in CLL cells. Honokiol induces caspase-dependent cell death of CLL cells, characterized by an increase in Annexin V positive, propidium iodide negative cells that was blocked by the caspase inhibitor, Z-VAD-fmk. Further evidence for Honokiol-induced apoptosis of CLL cells was confirmed by an increase in caspase 3 activity and cleavage of PARP. In addition, Honokiol demonstrated an LC50 that was lower for CLL cells than for normal mononuclear cells, suggesting that CLL cells are more susceptible to the cytotoxic effects of Honokiol compared to normal hematopoietic cells. To determine whether Honokiol-induced apoptosis of CLL cells was associated with known positive therapeutic effects, we examined mcl-1 expression, a survival protein whose down-regulation is associated with response to treatment in CLL patients. Honokiol treatment of CLL cells resulted in a significant decrease in the expression of mcl-1 within 24 hours. Furthermore, CLL cells pre-treated with IL-4, a cytokine known to support CLL survival, underwent apoptosis when subsequently incubated with Honokiol, indicating that Honokiol could also overcome the pro-survival effects of IL-4. These data indicate that Honokiol is a potent inducer of apoptosis in CLL cells and should be examined for further clinical application.
Telomere Length as a Prognostic Parameter in Chronic Lymphocytic Leukemia with Reference to IGV(H) Gene Mutation Status.
Authors: Roos Goran , Grabowski Pawel , Hultdin Magnus , Karlsson Karin , Thunberg Ulf , Tobin Gerard , Laurell Anna , Rosenquist Richard
Published: April 2020

Recent studies on the immunoglobulin variable heavy chain (IgV(H)) genes have revealed that B-cell chronic lymphocytic leukemia (B-CLL) consists of at least 2 entities with either somatically mutated or unmutated V(H) genes. Cases with unmutated V(H) genes, considered to evolve from pregerminal centre (GC) cells, have a worse outcome compared to cases showing mutated V(H) genes, that is, GC experienced or post-GC derived. Also, telomere length has been reported to be of prognostic significance in CLL. Telomerase becomes activated during the GC reaction with an elongation of the GC-B cell telomeres and a strong association exists between V(H) gene mutation status and telomere length in CLL (ie mutated cases have longer telomeres).

These results encouraged us to study telomere length and VH gene mutations in relation to clinical parameters in a large series of B-CLL cases (n=261). Telomere length was assessed by a PCR based method (Cawthon, 2002) showing very good correlation to telomere length data obtained by Southern blotting. Ig gene rearrangements were determined by DNA sequencing. The prognostic information given by the Ig mutation status (mutated vs unmutated) as well as by telomere length ("long" vs "short", cut off at median value) was statistically highly significant (p<0.0001 for both). Interestingly, the prognostic information given by telomere length seemed to be restricted to the Ig mutated CLL cases.

Our data indicate that telomere length must be considered as a relevant prognostic factor of importance for B-CLL. Telomere length determination using the here applied PCR technique is fast and accurate and can easily be established in routine diagnostics.
Motexafin Gadolinium, a Redox Mediator, Induces Apoptosis in Chronic Lymphocytic Leukemia B Cells and Is Additive or Synergistic with Fludarabine.
Authors: Naumovski Louie , Bone Nancy D. , Sirisawad Mint , Miller Richard , Jelinek Diane F. , Tschumper Renee C. , Kay Neil E.
Published: April 2020

Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that oxidizes intracellular reducing metabolites and protein thiols, thereby generating reactive oxygen species (ROS) through a process known as redox cycling. B-cell chronic lymphocytic leukemia (CLL) has elevated levels of the antioxidant ascorbate, and is known to be sensitive to redox stress. Since MGd can generate ROS through oxidation of ascorbate, we reasoned that MGd would be cytotoxic to CLL cells. Mononuclear cells were isolated from the blood of CLL patients (n=15) characterized by Rai Stage, CD38, FISH and immunoglobulin heavy chain variable (Ig VH) mutational status. CLL cells were incubated in vitro over 2–5 days with MGd at concentrations ranging from 0–50 uM. Cytotoxicity was assessed by flow cytometry using annexin V binding/propidium iodide uptake. Accumulation of MGd in CLL cells was demonstrated by flow cytometry using drug fluorescence (>650 nm). MGd-induced cytotoxicity was found to be dose-dependent over a range of 2.5 to 50 uM in cells from 11 of 15 CLL patients. CLL cells from 11/15 patients treated with 50 uM MGd and 4/15 patients treated with 10 uM MGd had cytotoxicity levels at least 2 fold above baseline. MGd uptake was similar in sensitive and resistant CLL cells, implying that mechanisms other than uptake are responsible for the differential responses. CLL cells that were sensitive to MGd treatment showed an increase in caspase-3 activity and cleavage of the caspase substrate PARP was found by immunoblot suggesting that MGd induced apoptosis. The induction of apoptosis was found to be exclusive of Rai stage, CLL FISH defects, CD38 expression or Ig VH mutational status. Cells from 5 CLL patients were studied for drug synergy using seven concentrations of MGd (7.5 – 50 uM) and the active moiety F-ara-A of fludarabine (0.25 –2.0 uM). The cells were treated with each drug individually or both drugs for 48 hours prior to analysis for cytotoxicity. MGd was found to exhibit additive activity (N=4) or synergistic (N=1) effects on CLL B cytotoxicity when combined with fludarabine. Thus, our pre-clinical results reveal that MGd is cytotoxic to CLL cells in vitro through induction of apoptosis. Furthermore, MGd shows additive or synergistic activity when combined with fludarabine. These results support the evaluation of MGd in ongoing CLL clinical trials utilizing this agent alone or in combination with fludarabine.
Effect of 2-Deoxyglucose, a Glycolytic Inhibitor, on the ATP Levels and Viability of B Lymphocytes from Subjects with Chronic Lymphocytic Leukemia (CLL).
Authors: Tidmarsh George F. , Tanner Laura I. , O’Connor Julie , Eng Clarence , Mordec Kasia
Published: April 2020

One hallmark of tumor cells is a high rate of glycolysis, which underlies the utility of the glucose analog, 2-[18]fluoro-2-deoxyglucose for PET imaging of solid tumors. The glucose analog, 2-deoxyglucose (2DG), inhibits glycolysis, and is predicted to decrease the viability of tumor cells, which are primarily dependent on the glycolytic pathway for ATP generation. We have recently shown that 2DG enhances the efficacy of either adriamycin or paclitaxel in delaying tumor growth in mouse xenograft cancer models (Maschek et al, Cancer Res 64 (2004): 31). We are currently conducting a phase 1 clinical trial to examine the safety of a combination administration of 2DG and Taxotere in patients with solid tumors. The goal of the current study is to measure the extent to which 2DG inhibits energy production and, consequently, reduces viability of leukemic cells. Heparinized blood was obtained from four subjects with a confirmed diagnosis of CLL (peripheral lymphocyte blood count ranged from 5,000–24,000/mm3), with an additional sample drawn from each subject two weeks later for replicate determinations. The mononuclear cell fraction (predominantly B cells) was isolated using Histopaque. Subsequent treatment of isolated CLL cells with 2DG (164 μg/ml) at 37°C (in the presence of 0.5 mM glucose) resulted in a 20–30% decrease in cellular ATP levels after 1 hr (compared with untreated cells) and a 60–70% decrease in ATP levels after 24 hrs. In the presence of 5 mM glucose, incubation of CLL cells with 2DG elicited a 5–10% decrease in ATP at 1 hr and 15–20% reduction in ATP levels at 24 hrs. The effect of 2DG on cell viability was assessed using Alamar Blue. Treatment of CLL cells with 2DG (164 μg/ml, in the presence of 0.5 mM glucose) resulted in a 50% loss in cell viability at 24 hrs (compared with untreated cells at the same time point), and a 70% loss at 48 hrs. In the presence of 5 mM glucose, the effect of 2DG was more modest. At 24 hrs, there was a 20% loss of cell viability and a 30–50% loss at 48 hrs. In conclusion, these results demonstrate that in vitro treatment of CLL B cells with 2DG decreases cellular ATP levels, ultimately resulting in a loss of cell viability. Updated results with samples from additional CLL subjects will be presented, together with an assessment of the therapeutic potential of 2DG for the treatment of CLL.
Treatment of Patients with Chronic Lymphoproliferative Disorders (CLPD) with Fludarabine, Cyclophosphamide, and Rituximab (FCR) Combination.
Authors: Galanopoulos Athanasios G. , Tsakiridou Anastasia , Michalis Eurydiki , Marinakis Theodoros , Gortzolidis George , Zomas Athanasios , Tsourveloudis Ioannis , Tsirogianni Maria , Papadhimitriou Stephanos , Kritikou-Griva Elpiniki , Anagnostopoulos Nikolaos I.
Published: April 2020

The FCR combination in a phase III study (Blood 102, abstract #373, p110a,2003) has shown promising results in the treatment of chronic lymphocytic leukemia (CLL) patients leading in increase of proportion of complete remissions and improved survival. Based on these encouraging results we tried to study the efficacy and safety of the FCR combination in patients of our Haematologic Unit. Seventeen patients, 8 males and 9 females with a median age of 69,5 years old, with relapsed/refractory or de novo CLPD ( 9 CLL and 8 NHL patients) were enrolled in this study between February 2002 and August 2004. Fifty percent of CLL patients had Rai stage I/II and the rest 50% had Rai stage III/IV disease. Four NHL patients had also Ann Arbor stage I/II and the rest four Ann Arbor stage III/IV disease. They were treated with Rituximab 375 mg/m2 on day1 in combination with Fludarabine and Cyclophosphamide ( 25 mg/m2 and 250 mg/m2 respectively) for days 2 to 4, every 4 weeks, for 6 consecutive cycles. Nine patients had a history of a prior unsuccessful treatment. Overall, 14 out of 17 evaluable patients were responsive to the treatment [12 patients complete response (CR) and 2 patients partial response (PR), overall response rate (ORR) 82%]. The remaining 3 patients did not show any response (NR), 2 progressive and 1 stable disease. Hematological toxicity was acceptable ( grade 2–3 neutropenia in 6/17 patients, grade 2–3 anemia and thrombocytopenia in 2/17 patients). There were no septic episodes except one case with neutropenic fever. There were no adverse events like nausea or vomiting except one patient with a serious anaphylactic reaction due to Rituximab administration. Three CLL patients died because of stable or progressive disease. In conclusion, this preliminary report suggests that the FCR regimen is an effective and safe treatment for CLPD patients, achieving higher CR rates than previous treatments. A longer follow up of a larger number of patients is required to confirm an improved survival in these patients.
FLT3/ITD Expression Increases Expansion, Survival and Entry into Cell Cycle of Human Hematopoietic Stem Cells.
Authors: Li Li , Piloto Obdulio , Kim Kyu-Tae , Ye Zhaohui , Nguyen Bao , Yu Xiaobing , Levis Mark , Cheng Linzhao , Small Donald
Published: April 2020

FMS-like tyrosine kinase-3 (FLT3) is a Class III receptor tyrosine kinase that is important for normal hematopoiesis. Activating mutations of FLT3 by internal tandem duplications (ITDs) in the juxtamembrane region are the most common molecular aberrations found in acute myeloid leukemia (AML). The contributions of FLT3 activating mutations in the leukemic transformation of normal human hematopoietic stem/progenitor cells (HSCs) have not yet been fully elucidated. In this study, using a single lentiviral vector containing two promoters, we achieved consistent and efficient coexpression of FLT3/ITD and green fluorescent protein (GFP) in transduced human CD34+ HSCs. When cultured in medium containing SCF, TPO and FLT3 ligand (FL), FLT3/ITD-transduced cells survived with enhanced self-renewal and survival potential, which was not affected by the withdrawal of FL. These cells retained a surface immunophenotype typical of HSCs (CD34+CD38−). Compared to cells transduced with a vector expressing GFP alone, FLT3/ITD-transduced HSCs had a higher fraction of cells in cell cycle. Clonogenic assays showed that FLT3/ITD-transduced HSCs produced fewer CFU-GM, implying that they were at least partially blocked in their ability to differentiate along the myeloid lineage. FLT3/ITD-transduced HSCs were more sensitive to the induction of cytotoxicity by CEP-701, a selective FLT3 inhibitor. In the FLT3/ITD-transduced HSCs, we detected increased expression of Pim-1, a serine/threonine kinase with an important role in cell survival, proliferation and differentiation, c-myc, a transcription factor involved in cell proliferation and cell cycle regulation, and Cyclin D3, a key factor in cell cycle regulation, each of which may contribute to the altered genetic program instituted by FLT3/ITD signaling.

These results together indicate that FLT3/ITD mutations may contribute to leukemic transformation of normal HSCs by prolonging survival, promoting proliferation, and blocking differentiation. CEP-701 may act as a potent agent for AML stem cells harboring FLT3/ITD mutations.
Cyclin D1 Immunohistochemistry Identifies a Subset of Plasma Cell Myeloma with Superior Overall Survival.
Authors: Cook James R. , Hsi Eric D. , Tubbs Raymond R. , Worley Sarah , Hussein Mohamad A.
Published: April 2020

The expression of Cyclin D1 is dysregulated in approximately half of cases of plasma cell myeloma due to translocations, aneusomy, or other abnormalities. Recent studies using quantitative mRNA analysis have suggested that increased Cyclin D1 mRNA expression is associated with a favorable prognosis. Previous attempts to examine the significance of cyclin D1 protein expression by immunohistochemistry have been hampered by the use of antibodies with weak staining and high background. In this study, we employ a newly available, commercial antibody that gives superior staining in B5 fixed tissues. We performed immunohistochemistry for Cyclin D1 on bone marrow core biopsies from a series of 44 newly diagnosed plasma cell myeloma patients who were uniformly treated on a Phase II study of rituxan, melphalan and prednisone. 22 patients (50%) were positive for Cyclin D1, defined as any plasma cells with positive nuclear staining. Cyclin D1 positive and negative cases displayed no significant differences in the initial levels of β2m (3.6±0.5 mg/L vs. 3.5±0.5 mg/L, p=0.860), number of bone marrow plasma cells (63±5.5% vs. 47±6.2%, p=0.063), or proportion of cases classified as SWOG stage 3-4 (2 of 22 (9%) vs. 5 of 22 (23%), p=0.412). The cyclin D1 positive cases displayed a superior overall survival with an estimated 3-year survival of 95% for Cyclin D1 positive cases versus 56% for Cyclin D1 negative cases (p=0.032). The cyclin D1 positive cases also displayed a trend towards better progression-free survival (median progression free survival of 15.7 months for Cyclin D1 positive versus 12.8 months for Cyclin D1 negative, p=0.13). In a Cox proportional hazards regression model, used to compare the effect of Cyclin D1 protein expression on overall survival time while adjusting for stage, the Cyclin D1 positive patients continued to show a strong trend to better overall survival (p=0.062). This study demonstrates that cyclin D1 immunohistochemistry, which could be readily performed in most pathology laboratories, is capable of identifying a subset of plasma cell myeloma with a favorable survival. Additional studies are ongoing to determine if these results can be generalized to other forms of therapy. If confirmed, routine cyclin D1 immunohistochemistry at the time of diagnosis may offer important prognostic information that could identify lower risk patients for whom less intensive therapies might be appropriate.
Ploidy, as Detected by Fluorescence In Situ Hybridization, Defines Different Subgroups in Multiple Myeloma.
Authors: Avet-Loiseau Herve , Wuilleme Soraya , Robillard Nelly , Lode Laurence , Magrangeas Florence , Beris Helen , Harousseau Jean-Luc , Proffitt John , Minvielle Stephane
Published: April 2020

Ploidy appears as an important parameter in both the biology and the clinical evolution of multiple myeloma. However, its evaluation requires either a successful karyotyping (obtained in 30% of the patients), or a DNA index calculation by flow cytometry (not routinely performed in myeloma). We validated a novel method for the detection of hyperdiploidy based on interphase fluorescence in situ hybridization (FISH) that can be utilized on almost all patients. We selected a 3-color probe set composed of probes specific for chromosomes 5, 9, and 15. Two hundred and five patients with myeloma were analyzed by flow cytometry (DNA Index) and FISH, and results obtained by both techniques were then compared. As shown in the table, FISH was very specific and sensitive for the detection of hyperdiploidy. Only 6 of the 104 hyperdiploid patients, all with a low hyperdiploidy (DNA Index <1.10), were classified as diploid by FISH. Extended studies looking for t(4;14), t(11;14), and del(13q14) showed that most recurrent 14q32 translocations occur in non-hyperdiploid clones, and that deletions of chromosome 13 were less frequently observed in hyperdiploid clones (48% vs 66%). These data confirmed that chromosomal abnormalities are not randomly distributed in myeloma and that FISH allows analysis of all the major chromosomal abnormalities. Further large studies are ongoing to evaluate the prognostic value of ploidy in myeloma and to correlate the true prognostic value of all chromosomal abnormalities.

Diploid by FISH Hyperdiploid by FISH Tetraploid by FISH t(11;14) t(4;14) del(13q14) DNA index < 1 7 3 2 3 DNA index=1–1.04 91 42 17 61 DNA index=1.05–1.5 6 98 7 7 50 DNA index >1.6 3 1 2
Expression of TRAIL Receptors on the Multiple Myeloma Cells.
Authors: Li Juan , Li Junhe , Luo Shaokai , Zhao Yin
Published: April 2020

Objective To study the different expression of death receptors and decoy receptors on mononuclear cells from patients with multiple myeloma and myeloma cell line KM3 and compare the different expression of TRAIL receptors after chemotherapy or exposure to doxorubicin, to explore the mechanisms by which TRAIL selectively kills tumor cells.

Methods Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry was used to investigate the expression of four receptors on mononuclear cells in 23 multiple myeloma patients and myeloma cell line KM3 and 15 controls, we furthermore compared the changes of expression mode after chemotherapy and incubation of KM3 cell with sub-clinical concentration of Doxorubicin.

Results There finds only DR4 and DR5 on KM3 cell line without the expression of DcR1 and DcR2. Expression of DR4 and DR5 on mononuclear cells of MM patients is higher than that of controls (P<0.05), but DcR1 and DcR2 expression was lower than that of controls (P<0.05), after chemotherapy and exposure to Doxorubicin, the expression of DR5 on MM cells was up-regulated (P<0.05)

Conclusions The expression of four receptors on myeloma cells and normal controls was significantly different, which might account for the selective killing effect of TRAIL on MM cells. DR5 was up-regulated on KM3 when incubating with Doxorubicin and after chemotherapy which suggests chemotherapy agents might enhance the apopotosis of MM cells through up-regulating of DR5 receptor.
Frequent Detection of Minimal Residual Disease (MRD) in Autografts from Patients with Multiple Myeloma during Remission Using a New Culture Method.
Authors: Cohen Yossi , Hardan Yzhar , Nagler Arnon , Zipori Dov
Published: April 2020

Current treatment of multiple myeloma includes autologous stem cell transplantation. However, it is unknown at the moment what is the extent of graft contamination with clonotypic myeloma cells. In order to evaluate the extent of residual contamination of the graft with myeloma cells, we used our new myeloma cell culture and expansion method developed in the Weizmann Institute of Science for the detection of MRD. We observed readily growing residual myeloma cells in 6 of seven cases, confirmed by clonal markers (FACS, PCR and FISH). However, there was some variability in the pattern of growth; one case of plasma cell leukemia and two cases with t(4;14) showed earlier and more pronounced growth, whereas one case with systemic amyloidosis and another case with MGUS failed to grow in this culture. We are currently arranging a multicenter study for further assessment of these findings and aim to answer the question whether the culture can distinguish between multiple myeloma and other plasma cell dyscrasias. Another goal is to correlate the pattern of in vitro growth of multiple myeloma cells, with clinical and chromosomal characteristics.

Figure Figure
Low Incidence of Deletions of ATM (ataxia-telangiectasia mutated) Gene in Patients (pts) with Multiple Myeloma (MM) as Detected by FISH.
Authors: Chanan-Khan Asher Alban , Miller Kena , McCarthy Philip , Panasiewicz Joanna M. , Block AnneMarie W.
Published: April 2020

Loss of function of ATM gene is associated with higher incidence of leukemia and lymphoma. ATM localized to 11q22.3-q23.1, is a tumor suppressor gene and plays an important role in cell cycle arrest, DNA repair mechanisms, and apoptosis. The incidence of deletion of ATM in MM remains unknown. Using a new commercially available fluorescently labeled DNA probe, we investigated the incidence of homozygous and heterozygous deletions of the ATM gene locus to explore its potential role in the pathogenesis of multiple myeloma (MM). Fifty-three MM pts were evaluated by fluorescence in situ hybridization (FISH) using the Vysis, Inc. LSI ATM (11q22.3) probe to assess the status of the ATM gene. LSI ATM (11q22.3) is a ~500 kb probe that hybridizes to the 11q22.3 region of chromosome 11 and spans the entire ~184 kb ataxia telangiectasia mutated (ATM) gene and several others. Probe copy number was assessed in each patient after analyzing at least 200 interphase nuclei. Only pts with> 10% bone marrow involvements with malignant plasma cells were analyzed. Heterozygous ATM deletion was noted in 2/53 (3.7%) patients. These patients had 82% and 20% involvement of bone marrow (BM) with malignant plasma cells. Twenty-eight of 53 (%) patients showed an extra signal suggestive of trisomy 11, while no abnormality (2 signals) was noted in 23/53 (%) pts. There was no significant difference in the stage, B2M or extent of marrow involvement among patient with or without aberration of ATM gene. Up to 15–20% of the pts with B-cell lymphoproliferative disorders (CLL, mantle cell and other NHL) are reported to have ATM deletion suggesting a possible role of ATM protein in the pathogenesis of at least a subset of these disorders. Even though MM is a B-cell disorder, in our observation the prevalence of ATM deletion in these pts is uncommon and thus unlikely to be a major factor in the pathogenesis of MM.
Rituximab in Anti-Mag Associated Neuropathy : A Monocentric Experience.
Authors: Delarue Richard , Deau Benedicte , Alyanakian Marie-Alexandra , Varet Bruno R. , Hermine Olivier
Published: April 2020

Anti-MAG associated neuropathy is a rare disorder characterized by a sensory-motor polyneuropathy in the context of monoclonal IgM, which activity is directed against myelin-associated glycoprotein on the surface of myelin sheaths. Treatments are usually ineffective. Most common therapy is based on alkylating agents such as Chlorambucil, which however only rarely improve neurological tests nor induce disappearance of monoclonal component. More recently, the benefit of monoclonal anti-CD20 antibody Rituximab was reported in case reports. Thus, we have performed since 2002 a prospective pilot study to assess the benefit of Rituximab in anti-MAG associated neuropathy. We report here the first four successive patients treated.

Patients and Methods : inclusion criteria were as follow : peripheral sensory-motor polyneuropathy with clinical and electrophysiological symptoms compatible with anti-MAG associated neuropathy; serum monoclonal IgM; serum anti-MAG reactivity; absence of other cause of peripheral neuropathy such as amyloidosis; absence of underlying condition contra-indicating Rituximab therapy. Patients received 4 courses of Rituximab (375 mg/m2) with a 7 days interval between two infusions. Usual premedication with corticosteroids and paracetamol were administered. Evaluation of clinical, biological and immunochemical efficiency was performed every three months.

Results : characteristics of the first four patients treated are as follow : 3 males and 1 female; median age : 65 years (57 – 87); median time between onset of symptoms and Rituximab therapy : 4 years (3 – 6). Previous treatments were Chlorambucil (3/4), corticosteroids (2/4), intravenous Ig (1/4), anthracyclin-containing regimen (1/4), plasmapheresis (1/4). All were ineffective. Rituximab was well tolerated by all patients. Median follow-up after treatment is 2 years (1 – 3). None of the patients exhibit improvement of clinical neurological symptoms. Electrophysiological evaluation show only a mild improvement in 1 patient. Moreover, no decrease in monoclonal component nor disappearance of anti-MAG reactivity of serum were observed in any cases. Interestingly, one patient exhibited later neurological and biological improvement after high dose Melphalan followed by autologous stem cell transplantation.

Conclusion : Rituximab monotherapy does not seem to improve neurological outcome of patients with anti-MAG polyneuropathy as well as is unable to clear serum from monoclonal IgM. Therefore, new approaches are needed, for example with high dose chemotherapy with stem cell support in selected patients.