bluuebunny/crossref_metadata_2025 huggingface dataset:
Implications of Gene and p53 Protein Alterations in Determining Progression of Chronic Myeloid Leukemia Phases.
Authors: Cavalcanti Geraldo Barroso , Pereira Eliane , Sheiner Marcos Antônio Mauricio , da Cunha Vasconcelos Flavia , Klumb Claudete Esteves , Maia Raquel Ciuvalschi
Published: October 2019

TP53 gene and p53 protein play an important role in the control of the cell cycle and DNA repair. Alterations in the TP53 gene and in the p53 protein expression have been considered an unfavorable prognostic factor for certain forms of human cancer. Mutations in the TP53 gene in patients with chronic myeloid leukemia (CML) were found in up to 30 %, specially among those in blastic crisis. At present, the only widely available technology that reliable detects and defines all mutations is DNA sequencing. However, the routine of sequencing the entire TP53 gene, in all suggestive cases of mutation, in the laboratorial routine is prohibitively costly, complex, and time consuming. To screening of those genetic alterations for p53 protein expression and TP53 gene abnormalities, in CML patients, flow cytometry (FC) and single strand conformation polymorphism of polymerase chain reaction products (PCR-SSCP) techniques are proposed. We report the results of an analysis of 72 blood samples from CML patients: 54 in chronic phase (33 in initial chronic phase (ICP) and 21 in late chronic phase (LCP)), 7 in accelerated phase (AP) and 11 in blastic crisis (BC). DNA structure for exons 5, 6, 7, 8 and 8–9 of the TP53 gene and p53 protein expression using the monoclonal antibody DO7 by FC was performed. By PCR-SSCP analysis, shift in eletrophoretic mobility of the TP53 gene, were detected in 11 patients and p53 protein were expressed in 17 out of 72 CML patients. The abnormal PCR-SSCP pattern were showed in one case of ICP (exon 7), five cases of LCP (two in the exon 7, one in the exon 8 and two in the exons 8–9), one in AP (exon 8) and four in BC (two in the exon 5, one in the exon 6 and one in the exon 8–9). In these cases, the p53 protein were expressed in seven cases (in all cases of CB and AC and 2 out of 5 LCP samples of CML patients). The statistic analysis by qui-square test showed significance between the results of the two methods (p= 0,002). These results suggest that the two concomitant methods can increase the sensibility for screening the p53 protein expression and TP53 gene abnormalities in CML patients. The presence of abnormal profile of PCR-SSCP associated to the p53 protein expression, in advanced phases of this disease, might be considered an important indicator of the phase progression of CML.
Effects of Arsenic Trioxide on Protein Kinase C and Cytosolic Calcium in Ventriclar Myocardium of Guinea Pig.
Authors: Zhou Jin , Meng Ran , Meng Lu , Yang Baofeng
Published: October 2019

AIM To clarify the mechanisms of the cardiac side effects of arsenic trioxide (As2O3) in leukemia treatment, the effects of As2O3 on protein kinase C(PKC) and cytosolic calcium ([Ca2+]i) in ventriclar myocardium of guinea pig were investigated.

METHODS Fluo-3/AM fluorescent probe tagged [Ca2+]i of guinea pig’s ventriclar myocardium, detected the [Ca2+]i, by laser confocal microscopy before and after exposed to As2O3. Phosphorus radioisotope assay was used to detect the activity of PKC on cell membrane or in cytoplasm. DNA ladders of ventriclar myocardium in Tyrode’s solution after exposed to As2O3 were evaluated.

RESULTS The cytosolic [Ca2+]i did not change when incubated guinea pig’s ventriclar myocardium in Tyrode’s solution with 0.5μmol•L−1As2O3, after incubated in2μmol•L−1As2O3 for 6min, [Ca2+]i began to increase and was continuous increasing for about 9min. After exposed to 10μmol•L−1As2O3 for less than 3 min, the [Ca2+]i kept on increasing till the end (about 27min) of the examination and could not recover. The increasing of [Ca2+]i could be partly blocked by calcium channel blocking agents—verapamil and nifedipin. The PKC of myocardial cells began to rise after incubated in an As2O3 changing concentration culture media for 3 hours, which initial As2O3 was 10μmol•L−1, and the DNA ladders appeared after incubated in it for10 hours. No DNA ladders appeared when incubated in an As2O3 culture media in which the initial As2O3 concentration was under 5μmol•L−1.

CONCLUSIONS As2O3 can active PKC, increase cytosolic [Ca2+]i and promote apoptosis of myocardial cells. The promoting apoptosis action of As2O3 is closely related to the lasting activation of PKC. The initial action target of As2O3 is cell membrane.
Imatinib (STI571) Induces DNA Damage in BCR/ABL-Expressing Leukemic Cells but Not in Normal Lymphocytes.
Authors: Blasiak Janusz , Drzewoski Jozef , Poplawski Tomasz , Czechowska Agnieszka
Published: October 2019

Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets specifically the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.05 to 2 μM induced DNA damage in human leukemic K562 cells expressing the BCR/ABL oncogene, whereas it had no effect in normal human lymphocytes. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the plasmid relaxation assay. K562 cells were unable to repair H2O2-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes.
Suppression of MLL-AF4 by Small Interfering RNAs Inhibits Proliferation and Induces Apoptosis in T(4;11)-Positive SEM Cells.
Authors: Greil Johann , Thomas Maria , Huenefeld Christian , Vornlocher Hans-Peter , Hadwiger Philipp , Heidenreich Olaf
Published: October 2019

The reciprocal chromosomal translocation t(4;11)(q21;q23) creates the fusion genes MLL-AF4 and AF4-MLL located on derivative chromosome 11 or derivative chromosome 4, respectively. We used small interfering RNAs to suppress the MLL-AF4 fusion gene in the t(4;11)-positive leukaemic cell line SEM. Electroporation of SEM cells with MLL-AF4 siRNAs caused a more than two fold transient decrease in MLL-AF4 mRNA levels, which lasted for three days. The reduction in MLL-AF4 fusion transcript levels were associated with a severely diminished clonogenicity, inhibition of proliferation and of G1-S cell cycle transition and induction of apoptosis. Therefore, MLL-AF4 siRNAs are not only useful to study the functions of MLL-AF4 in leukaemogenesis, but may be also promising agents for novel treatment concepts for t(4;11)-associated leukaemias.
Effects of Protein Tyrosine Kinase, Protein Tyrosine Phosphatase and Protein Kinase C on the Apoptotic Process of Leukemia Cells and Human Cortex Neurons Managed by Arsenic Trioxide.
Authors: Zhou Jin , Meng Ran , Yang Baofeng , Li Baoxin
Published: October 2019

Objective Study on the effects of protein tyrosine kinase, protein tyrosine phosphatase and protein kinase C (PKC)on apoptotic process, observe the changes of cytosolic calcium ([Ca2+]i) of human leukemia cells and cortex neurons after clinical relevant dosage of arsenic trioxide (As2O3) exposure in vitro.

Methods Marked [Ca2+]I of human leukemia cells and cortex neurons with Fluo-3/AM, assayed the changes of [Ca2+]I with laser confocal microscopy in real-time after managed with As2O3 in different concentrations respectively, detected the effects of protein tyrosine kinase, the protein tyrosine phosphatase and the activation of protein kinase C on these changes with confocal microscopy and Phosphorus radioisotope assay. Assayed the DNA ladders of leukemia cells and cortex neurons after exposed in As2O3.

Results As2O3 1μmol/L can increase the [Ca2+]I of NB4 cells remarkably but show no effects on neurons. Vanadate, a kind of protein tyrosine phosphatase inhibitor, can dose-dependently promote the increasing of [Ca2+]I caused by 2, 5, 10μmol/L As2O3.The mean value of total increased rate in 280 seconds after exposed in As2O3 in different concentrations were: 6.5±2.3%, 21.7±2.1 %,49.2±2.5% in NB4 cells, and 6.7±2.1%, 19.4±2.5 %, 52.3±2.7% in cortex neurons respectively. Genistein, a kind of protein tyrosine kinase inhibitor, can dose-dependently decrease the raising of [Ca2+]I caused by 2, 5, 10μmol/L As2O3.The mean value of total inhibited rate in 280 seconds after As2O3 management in different concentrations were 6.7±2.9%, 25.6±2.5 %, 52.2±3.5% in NB4 cells, and 7.8±3.1%, 18.1±2.8%, 51.3±3.3% in cortex neurons respectively. The activation of PKC began when exposed in As2O31μmol/L for 3 h, and keep raising continuously in NB4 cells., DNA ladders appeared after exposed in As2O31μmol/L for 24h. However, no appearances above were found in human cortex neurons. In 2μmol/L As2O3 exposedness, The activation of PKC and DNA ladders appeared in neurons.

Conclusions The phosphorylation and dephosphorylation of protein tyrosine kinase and protein tyrosine phosphatase participated in nonspecific apoptosis signal transduction pathway of As2O3, and accompanied with PKC activation. The [Ca2+] elevation was closely related to PKC increasing activation. The tolerances between leukemia cell and cortex neuron to As2O3 were differ in dosage.
Molecular Detection of Bone Marrow (BM) Involvement in Primary Cutaneous B Cell Non Hodgkin’s Lymphoma (NHL) at Diagnosis.
Authors: Gameiro Paula , Cabeçadas José , Sebastião Marta , Sachse Fernanda , Nolasco Inês D. , Lúcio Paulo J. , Silva Maria G.
Published: October 2019

Although the skin is the second most common site of involvement for extranodal lymphomas, only 20% of cutaneous NHL are of B cell origin. A wide range of B cell lymphomas can occur as primary cutaneous tumors and their clinical course can not always be predicted by histology alone. The incidence and significance of the presence of clonal B cell populations in the BM of affected patients at the time of diagnosis are presently unknown. On another hand, the BIOMED II primers were shown to have a high sensitivity for the detection of clonality in B cell lymphoproliferative disorders. We sought to determine the incidence of occult BM involvement in a series of twelve patients (3 women and 9 men, median age 54.5, 28 to 77 years old) diagnosed with primary cutaneous B cell lymphoma between August 2000 and May 2004, for whom skin biopsies, BM trephine biopsies and aspirates and clinical data were available for review. For that, we investigated the presence of clonal B cell populations in the BM aspirates obtained as part of the initial staging procedures by PCR (employing heteroduplex and GeneScanning analysis), using the BIOMED II primers for the immunoglobulin k light chain (IGK) and the immunoglobulin heavy chain complete and incomplete (IGH) rearrangements. At the same time, a set of amplified gene fragments of known sizes was run as a control of the DNA integrity. Patients’ diagnoses, according to the WHO classification, were marginal zone B cell lymphoma (5 cases), diffuse large B cell lymphoma (5 cases), follicular lymphoma (1 case) and NHL NOS (1 case). In all cases clinical evaluation, CT scans from the thorax, abdomen and pelvis as well as BM trephine biopsies failed to demonstrate extracutaneous involvement by NHL. Moreover, a maximum of 6% of policlonal CD19+ B cells were present in the BM aspirates as evaluated by flow cytometry. In three out of 12 cases (25%) a B cell clonal population was present in the BM, as demonstrated by the presence of an IGH clonal rearrangement detected by heteroduplex and/or GeneScanning analysis; they corresponded to two diffuse large B cell NHL cases and one marginal zone B cell lymphoma. Two of these patients are still under treatment and one (with diffuse large B cell lymphoma) remains in complete remission 9 months after first line therapy. In this small series of patients the detection of clonal B cell rearrangements in the BM analyzed at the time of diagnosis was uncommon; the clinical course of the positive cases did not differ from the whole series. Further follow-up studies are needed to define the significance and prognostic impact of those BM clones in cutaneous B cell NHL patients.
Prognostic Relevance of Gene Expression Using RT-PCR in Diffuse Large B-Cell Lymphoma.
Authors: Kim Hee-Nam , Lee Je-Jung , Yang Deok-Hwan , Kim Yeo-Kyeoung , Kim Nan Young , Park Kyeong-Soo , Kim Hyo-Jung , Jang Dae-Young , Ahn Jin-Seok , Bae Sung-Hwa , Ryoo Hun-Mo , Kim Hyeoung-Joon
Published: October 2019

Diffuse large B-cell lymphoma (DLBCL) is characterized by a marked degree of morphologic and clinical heterogeneity. Recently, Rosenwalt et al. (N Engl J Med 2002) reported that four gene expression “signature”, 17 genes were identified as correlated with patient outcome by DNA microarray in DLBCL. In this study, we aim to establish predictor of outcome could help to identify patients who may benefit from risk-adjusted therapies in advance. To do it, we evaluate the prognostic relevance of 17 gene expressions in 72 patients with DLBCL who received a conventional chemotherapy. Seventeen genes were studied using RT-PCR assay from paraffin-embedded sections at the time of diagnosis. The median age of the patients was 58 years (range: 21–80 years). When we initially exam an appropriative patient’s selection for survival analysis, overall survival (OS) at 2 years in patients with the international prognostic index (IPI) < 2 and IPI ≥ 2 were 95.2±4.6% and 50.6±11.8%, respectively (p = 0.009), and progression free survival (PFS) at 2 years in patients with the IPI < 2 and IPI ≥ 2 were 75.0±9.7% and 46.7±12.9%, respectively (p = 0.049). Of the 17 genes, patients with uPA expression showed a shorter OS compared with those without the gene expression. Additionally, patients with the expression of NPM3, uPA, fibronectin, or IMAGE814622 showed a shorter PFS compared with those without the gene expressions. In conclusion, these findings suggest that the gene expression profiling with simple RT-PCR assay is useful for analysis of the prognostic implications in patients with DLBCL.
Bim-EL, Is Sensitive to Proteasome Degradation and Contributes to Inducing Apoptosis after In Vitro Imatinib Mesylate Treatment of BCR-ABL Positive Cells.
Authors: Belloc Francis , Boyer Catherine , Lacombe Francis , Praloran Vincent , Mahon François-Xavier
Published: October 2019

Chronic myeloid leukemia (CML) is a malignant disease of pluripotent hematopoietic stem cells and is characterized by excessive accumulation of the myeloid lineage. The causative event in the initiation of CML is the translocation of the abl gene in continuity with the bcr gene resulting in a fusion gene encoding for a Bcr-Abl tyrosine kinase. The expression of Bcr-Abl protects hematopoietic cells against apoptosis. We investigated the survival pathways triggered by Bcr-Abl. Apoptosis was measured in two cell lines expressing the Bcr-Abl protein either constitutively (K562) or ectopically (BaF/Bcr-Abl). Imatinib mesylate, an inhibitor of the tyrosine kinase activity of Bcr-Abl, induced apoptosis and caspase activation in K562 and BaF/Bcr-Abl and this effect was almost completely inhibited by cycloheximide (CHX) indicating that imatinib needs de novo protein synthesis to induce apoptosis in Bcr-Abl cell lines. MG132, a proteasome inhibitor, was also found to induce apoptosis in both cell lines, an effect also inhibited by CHX. By measuring mitochondrial membrane potential we found that CHX affected the apoptotic process induced by both inhibitors upstream of the mitochondrial step. Co-treatment with imatinib and MG132 failed to show any additive effect and imatinib did not inhibit proteasome activity by itself. These results are in agreement with Bcr-Abl tyrosine kinase exerting its anti-apoptotic effect by driving the degradation of a pro-apoptotic protein with a high turnover which, after phosphorylation, became a substrate for the proteasome. When protein kinase C was activated by PMA, Bim-EL, a pro-apoptotic, BH3-only member of the Bcl-2 family, was converted in its phosphorylated form and its cell content was decreased by 60%. The phosphorylated form of Bim-EL was preserved when proteasome was inhibited by MG132 inducing a two times increase in Bim-EL protein content. Similarly, the content in Bim-EL was increased (three times in K562 and five times in BaF/Bcr-Abl) after 18h imatinib treatment. In this case, Bim-EL accumulated in an un-phosphorylated form and this accumulation was inhibited by CHX. Thus, Bcr-Abl kinase could indirectly drive the phosphorylation of Bim-EL, maintaining its degradation by the proteasome and, consequently, a low apoptotic potential for the cell. Conversely, Bim-EL accumulation could play a crucial role in the apoptotic response of Bcr-Abl expressing cells treated by either imatinib or proteasome inhibitors.
Association of Hydroxyurea to Imatinib Is Effective in Patients with Chronic Myelogenous Leukemia Resistant to Imatinib Alone.
Authors: Latagliata Roberto , Breccia Massimo , Carmosino Ida , Sarlo Chiara , De Cuia Rosa , Mancini Marco , Biondo Francesca , Russo Eleonora , De Vellis Annalisa , Diverio Daniela , Mandelli Franco , Alimena Giuliana
Published: October 2019

The introduction of Imatinib in the treatment of Chronic Myelogenous Leukemia (CML) leads to the achievement of Complete Cytogenetic Response (CCR) in about 70% of patients: however, in the remaining 30% of patients there is a persistance of Ph+ cells also after standard (400 mg/day) and increased (600 mg/day) dose of Imatinib. These patients are thus cytogenetically resistant to Imatinib alone and their management is at present unclear. From 11/2002 to 11/2003, 10 patients in chronic phase (6 male and 4 female, median age 52.5 years, range 29 – 68 years) with persistance of 100% Ph+ cells (9 patients) or BCR/ABL + cells (1 patient with Ph- BCR/ABL+ CML at onset) after standard (at least 6 months of treatment) followed by increased dose (at least 3 months of treatment) of Imatinib alone, were considered resistant and added Hydroxyurea (HU) to Imatinib. Seven patients have been pretreated with IFN before Imatinib; median times from diagnosis and from Imatinib treatment to HU addition were 51 months (range 23 – 151) and 14 months (range 10 – 31), respectively. HU was given according to WBC count: patients with WBC < 10 x 109/l started HU at the dose of 1 g/day, patients with WBC > 10 x 109/l at the dose of 1.5 g/day. Imatinib was continued at the same previous dosage (600 mg/day in 6 patients and 400 mg/day in 4 patients who did not tolerate increased dosage for hematological toxicity). Three patients achieved a complete response (2 CCR after 3 and 12 months respectively and 1 molecular complete response after 9 months in the patient Ph- BCR/ABL+ at onset) and 1 patient achieved a partial CR (Ph+ < 33%) after 9 months: the remaining 6 patients were resistant with persistance of 100% Ph+ cells. Toxicity was mild and only 1 patient discontinued for 2 weeks the association due to transient thrombocytopenia: no extra-hematological toxicity has been recorded. After a median follow-up of 14 months (range 20 – 10), 2 patients (1 resistant and 1 after 5 months from the achievement of CCR) evolved in Blastic Phase (BP), 5 patients are in stable chronic phase with 100% Ph+ cells and 3 patients are still in response after 4,6 and 7 months respectively. In conclusion, the association of HU with Imatinib seems capable to induce cytogenetic response in at least one third of patients resistant to Imatinib alone, with minimal toxicity: a longer follow-up and a comparison with other associations is needed to evaluate the quality and duration of response in such group of patients.
Plasma Adiponectin Concentrations in Relation to Chronic Lymphocytic Leukemia and Chronic Myeloproliferative Diseases.
Authors: Avcu Ferit , Ural Ali U. , Yilmaz Mahmut I. , Bingol Necati , Nevruz Oral , Caglar Kayser
Published: October 2019

Adiponectin, an adipocyte-secreted hormone, is closely and inversely associated with insulin resistance. Adiponectin suppresses the expression of adhesion molecules in vascular endothelial cells and cytokine production from monocytes/macrophages, thus inhibiting the inflammatory processes. In addition, it is an important negative regulator in immune systems and hematopoiesis that suppressed colony formation from colony forming unit granulocyte and macrophage. For these immunological effects of adiponectin, we hypothesized that decreased adiponectin levels might underlie the association between chronic lymphocytic leukemia (CLL) and chronic myeloproliferative diseases (CMPD), and investigated the levels of adiponectin in patients with 19 CLL and 30 CMPD (14 chronic myelogenous leukemia, 9 polycythemia vera, 4 myelofibrosis, 2 essential thrombocythemia), and compared with age, sex, body mass index match controls. Adiponectin levels in patients with CLL were significantly lower than the controls (4.71±1.33 μg/ml vs. 16.61±3.91 μg/ml; p<0.001). Also, the levels of adiponectin in patients with CMPD were significantly lower than the controls (8.95±2.64μg/ml vs. 17.16±4.77 μg/ml; p<0.001). According to the comparison of interferon (IFN) therapy and non-IFN therapy of the patients with CMPD, plasma adiponectin levels in IFN therapy patients were significantly higher than in non-IFN therapy patients (11.03±1.39μg/ml vs. 6.87±1.79 μg/ml; p<0.001). The results show that adiponectin is lower in patients with both CML and CMPD. These results suggest that lymphopoiesis and myelopoiesis is negatively influence of adiponectin level and its possible role may be related with inflammatory cytokine (eg: TNF, IL-6) release. Also, IFN therapy that suppressed of inflammatory cytokines in patients with CMPD is positively influenced of adiponectin secretion. Future studies are needed to prove causality and provide insight on the mechanism of action of this hormone and its potential role in CML and CMPD.
B-Chronic Lymphocytic Leukemia (B-CLL), Small Lymphocytic Lymphoma (SLL) and Waldenstrom’s Macroglobulinemia (MW): A Comparative Fish Analysis.
Authors: Dimopoulou Maria N. , Angelopoulou Maria K. , Vassilakopoulos Theodoros P. , Kyrtsonis Marie-Christine , Koumbis Dafni , Kontopidou Flora N. , Siakantaris Marina P. , Kalpadakis Christine , Kokoris Styliani I. , Dimitriadou Evangelia , Pangalis Gerassimos A.
Published: October 2019

Chromosomal abnormalities in B-CLL are considered as potent prognostic factors. In other small lymphocytic disorders, closely related to B-CLL, such as SLL and MW the cytogenetic profile has not been extensively studied. The purpose of the present study is to identify any potential differences in chromosomal abnormalities between B-CLL, SLL and MW. We studied 24 consecutive B-CLL, 13 SLL and 8 MW patients diagnosed in our Unit. Diagnosis was based on standard morphologic and immunophenotypic criteria for B-CLL and SLL patients. Cases with absolute lymphocyte count < 5x 109/L were considered as SLL. The diagnosis of MW was established in the presence of serum monoclonal IgM and lymphocytic/lymphoplasmacytic infiltration of the bone marrow. Separated lymphocytes from peripheral blood (B-CLL) or bone marrow (SLL and MW) were fixed and FISH analysis for trisomy 12 and 13q deletion was performed according to standard techniques. 200 intact interphase nuclei were scored for each sample. Cut-off levels were defined by the mean value plus 3 standard deviations of the frequency of the abnormalities in 10 normal controls. 5/24 B-CLL cases were positive for trisomy 12 (21%) versus 6/12 (50%) SLL and 0/7 (0%) MW cases. Del 13q was found in 8/14 (57%) of B-CLL, versus 1/6 (17%) of SLL and 0/8 (0%) of MW patients. We found a difference in the cytogenetic profile between B-CLL and SLL, which represent two closely related entities with different tissue localization pattern. Thus trisomy 12 was more frequently observed in SLL, whereas the opposite was true for del 13q. In addition none of these two chromosomal abnormalities was encountered in MW, a finding that could potentially serve as an additional tool for the differential diagnosis between these related disorders. These findings need to be confirmed in larger series of patients and their biologic significance should be further investigated.
Extremely Peculiar Nuclear Changes in Cultured Primary Myeloma Cells, Including Bulging and Duplication/Multiplication of Nuclei and Cell Expansion without Apparent Mitotic Forms.
Authors: Cohen Yossi , Zipori Dov
Published: October 2019

Using our new cytokine-free, mitogen-free in vitro culture system for primary multiple myeloma cells (see abstract by Cohen Y and Zipori D), we have observed peculiar nuclear changes occurring during the first days in culture. After 3–4 days small lymphocytic-like cells are frequently seen, accompanied by typical plasma cells together with plasma-like cells possessing a single bulge in one edge of the nucleus (Figure 1). This nuclear bulging has been seen only in cultures from primary myeloma cases therefore it might represent a pathognomonic disease related phenomenon. Later in culture, multiple cells show nuclear bulging of variable shapes and degrees. It seems that this heterogeneity represents a spectrum of the same nuclear process (Figure 2). In addition, binucleated cells with eccentric nuclei are commonly seen at this stage, whereas multinucleated cells can be seen occasionally. Another observation is an apparent burrier-like structure between those nuclei in binucleated cells. Most interestingly, despite extensive expansion of the cultured myeloma cells (whose clonality was confirmed by FACS analysis, PCR and FISH-Bioview), mitotic forms are very rare. Furthermore, by combined FISH/morphology analysis of some cultured binucleated myeloma cells from a patient with del 13, a single signal was seen in each of the two nuclei using a probe for chromosome 13. Altogether, these unusual myeloma cell forms can now explain the yet unexplained finding frequently seen by FACS analysis of nuclear hyperploidity in myeloma cells. Currently, we use time lapse cinematography to assess the possibility that cultured myeloma cells undergo nuclear replication, through budding, and examine the possibility that these changes lead to the emergence of two daughter cells.

Figure Figure
Thalidomide-Induced Peropheral Neuropathy in Newly Diagnosed and Pre-Treated Multiple Myeloma Patients.
Authors: Tosi Patrizia , Zamagni Elena , Cellini Claudia , Cangini Delia , Tacchetti Paola , Perrone Giulia , Tura Sante , Baccarani Michele , Cavo Michele
Published: October 2019

Thalidomide is remarkably active in advanced relapsed and refractory multiple myeloma (MM), so that its use has been recently proposed either in newly diagnosed patients or as maintenance treatment after conventional or high-dose therapy. This latter therapeutic approach has risen the concern of side effects occurring after long-term therapy with this drug. In order to investigate this issue we analyzed the outcome of 74 patients who have been treated with thalidomide + dexamethasone for longer than 8 months at our Institution. Thirty-four patients (18M, 16F, median age = 55 years) had newly diagnosed symptomatic MM and were treated with four monthly courses of thalidomide 200mg/day + dexamethasone 40 mg on days 1 to 4 (even cycles), or on days 1 to 4, 9 to 12 and 17 to 20 (odd cycles), followed by cyclophosphamide 7g/m2 + G-CSF, peripheral blood stem cell (PBSC) collection, and double autologous PBSC transplant. Thalidomide + dexamethasone was administered throughout the whole treatment program. Forty patients (27M, 13F, median age = 61 years) were treated with thalidomide 200mg/day +dexamehasone 40mg on days 1–4 every four weeks as salvage therapy for relapsed (n = 14) or progressive (n=26) MM. Neurotoxicity was the most troublesome and frequent toxic effect that was observed after > 8 months treatment, the incidence averaging 74% in newly diagnosed patients and 75% in pretreated ones. Symptoms included paresthesias, tremor and dizziness; serial electromyographic studies revealed a symmetrical, mainly sensory peripheral neuropathy, with minor motor involvement. The severity of neuropathy, graded according the NCI-CTC 2.0 scoring system, varied greatly in the two groups of patients, as pretreated patients showed grade 2 and 3 toxicity in 32.5% and 27.5% of the cases, respectively, while the majority of newly diagnosed patients complained about grade 1 toxicity (57%), and none of them experienced grade 3 toxicity. In both groups thalidomide neurotoxicity was not related to sex, M protein isotype, and thalidomide daily dose. In pretreated patients, a significant correlation between grade 2 + 3 neurotoxicty and longer disease duration was found, thus suggesting that subclinical MM-related neurotoxicity could favour drug-induced toxic effects. These results suggest that long-term thalidomide therapy in MM may be hampered by the remarkable neurotoxicity of the drug, and that a neurological evaluation should be mandatory prior to thalidomide treatment, in order to identify patients at risk of developing a periperal neuropathy.
Clinical Responses to Sildenafil in Waldenstrom’s Macroglobulinemia.
Authors: Treon Steven P. , Tournilhac Olivier , Branagan Andrew , Hunter Zachary , Xu Lian , Hatjiharissi Evdoxia , Santos Daniel Ditzel
Published: October 2019

Waldenstrom’s macroglobulinemia (WM) is an incurable B-cell malignancy. Interestingly, we have observed unusual response activity in five WM patients which appears related to their use of sildenafil, a phosphodiesterase inhibitor used to treat erectile dysfunction. One patient demonstrated a complete remission, while four other patients demonstrated less dramatic, but also unexpected responses associated with sildenafil used at doses of 25–50 mg once to twice a week. In view of these observations, we next evaluated sildenafil for its ability to induce apoptosis of lymphoplasmacytic cells obtained from WM patients. All patients provided written consent for this study which was approved by our institutional review board. Sorted (CD19+, light chain restricted) bone marrow lymphoplasmacytic cells obtained from 6 WM patients were cultured in media alone or with sildenafil (kindly provided by Pfizer Pharmaceuticals, Inc.) at 0.01 ug/ml for 24 hours. These studies demonstrated a mean 2.1 (range 1.24–4.8) fold increase in sildenafil specific/spontaneous apoptosis in lymphoplasmacytic cells from 5 of 5 patients, which included those from a patient who demonstrated a clinical response to sildenafil. Importantly, the concentrations at which apoptosis was observed in these studies is within pharmacologically achievable levels for sildenafil. The results of these studies, along with those recently reported by Sarfati et al (Blood 101:265) who demonstrated caspase mediated apoptosis of B-chronic lymphocytic leukemia (CLL) cells by sildenafil, provide the framework for investigation of sildenafil and related phosphodiesterase inhibitors in the treatment of WM and other B-cell malignancies.
Intermittent Low Doses of Thalidomide in the Maintenance Treatment of Multiple Myeloma.
Authors: Bibas Michele , Andriani Alessandro , Viva Felicetta , Gualandi Carla , Mignogna Teresa , Geraldini Claudio , Tarquini Daniela
Published: October 2019

BACKGROUND: Despite recent advances multiple myeloma remains an incurable plasma cell malignancy. Thalidomide has recently been recognized as an effective agent for previously untreated, refractory or relapsed myeloma.

DESIGN AND METHODS: Thirty patients with refractory/relapsed MM (22 females, 8 males) aged from 53 to 81yrs : 18 MM IgG(k), 3 MM IgA(k), 6 MM IgG(l), 2 MM IgA(l) an MM (l) were treated with thalidomide in combination with high doses (40 mg monthly) of dexamethasone and monthly zolendronate( 4 mg).All patients were treated with conventional escalated dose of thalidomide every 2 weeks starting from 100 mg daily to max tolerated dose. Of the 30 patients 19 (63 %) responded to therapy with a monoclonal reduction of more than 50% ( 4 acheived CR). Among the responders those with polineurophaty documented as a subjective simptom and with electrophysiologic objective parameters alteration entered in a reduced thalidomide schedule of 100 mg daily 10 days a month. The follow up of these patients actually ranges between 2 to 21 months, 1 patient progressed and 2 patients discontinued treatment because of a subjective progression of neurological simptoms but without electrophysiologic alterations.

RESULTS: None of the patients treated with intermittent low doses of Thalidomide developped an increase of neurophaty. Intermittent low doses of THAL may be applicable in the maintenance treatment of myeloma patients.

Patients status

Response Responders Maintenance In Study Progressed Stopped PR 15 (50%) 6 3 1 (4 mo) 2 (3 and 6 mo) CR 4 (13 %) 2 2 0 0
Fluvastatin-Zometa (FluZom) as A Possible Therapeutic Approach to Plasma Cell Disorders.
Authors: Spencer Andrew , Baulch-Brown Cindy , Reed Kate , Lynch Kevin
Published: October 2019

Despite significant improvement in the outlook for newly diagnosed patients with multiple myeloma (MM) the disease remains incurable. Novel therapeutic approaches are therefore required. We have previously demonstrated in vitro that both the third generation nitrogen-containing bisphosphonate zometa and the HMG-CoA reductase inhibitor fluvastatin have anti-proliferative and apoptosis-inducing effects against human myeloma cell lines (HMCL). Furthermore, isobologram analyses with the 2 compounds have demonstrated synergistic activity against HMCL. Based on this we have undertaken a pilot study of fluvastatin and zometa (FluZom) in a group of patients with progressive plasma cell disorders (PCD). A 16-week treatment phase was planned with patients commencing fluvastatin 40mgdaily on day 1 escalating to 80mg daily from day 15 to day 112. Zometa 4mg IV over 15 minutes was given on days 29, 57, 85 and 112. Any patient demonstrating any response, based on conventional criteria, or stable disease (SD) by day 112 remained on treatment until further progression (PD). PD prior to day 112 mandated treatment withdrawal. Eleven patients were enrolled on trial - progressive MM (n = 9), smouldering myeloma (SMM) progressing to MM (n = 1) and progressive POEMS disease with multisystem involvement (n = 1). All except the SMM patient were heavily pretreated - median 3 (range, 0 – 6) prior treatment regimens, 7 with prior ASCT and 4 with prior radiotherapy. Median age was 61 years (range, 44 – 74). Six patients completed 16 weeks of therapy, 5 progressed prior. Of the former, 4 (36%) demonstrated SD and continued on therapy to a total of 20, 23 (patient no. 2), 28 (patient no.1) and 45 weeks. No patient demonstrated a disease response based on standard criteria. Patients no.’s 1 and 2 were pancytopenic prior to therapy and demonstrated normalisation of haemoglobin (no. 1), platelets (no. 1) or total leucocytes (no.’s 1 and 2). Two patients, neither of whom achieved SD but who completed 16 weeks of therapy, had a plasma cell labelling index (PCLI) undertaken pre and post therapy, both showed a reduction in the PCLI following therapy - 5.4% to 3.6% and 2.6% to 1.3%, respectively. Therapy was well tolerated with 3 patients developing transient elevation of liver transaminases. We conclude that FluZom while not producing disease response as judged by conventional criteria did nonetheless exhibit beneficial biological activity in a minority of this cohort of progressive PCD patients. Further evaluation in patients with early and/or untreated disease is warranted.
Complex Alterations in Serum Cytokines and Increased Il-4 Levels Characterize Patients Developing Chronic Thrombocytopenia after Allogeneic Hematopoietic Cell Transplantation.
Authors: Elshal M. , Takahashi Y. , Chakrabarti S. , Srinivasan R. , Suffredini D. , Igarashi T. , Solomon S. , Lundqvist A. , Barrett A. J. , McCoy J. P. , Childs Richard
Published: October 2019

Isolated chronic thrombocytopenia (CT) following allogeneic HCT is associated with an increased risk of transplant-related mortality and a reduction in long-term survival. Although the pathogenesis of CT is not entirely clear, it has been speculated that immune activation and cytokine dysregulation characteristic of chronic GVHD (CGVHD) play a similar role in the pathogenesis of this disorder. To better understand the impact of endogenous cytokine expression on platelets, we analyzed pro and anti-inflammatory cytokines in the serum of patients with CT following allogeneic HCT, comparing levels to healthy donors and to patients with normal platelet counts after allo-transplantation. Serum was obtained from 3 patient groups; Group 1- healthy donor controls (n=12): Group 2- post-transplant from allogeneic HCT patients with normal platelet counts (n=31): Group 3- post transplant from patients with CT (n=11). Using micro sphere-based Luminex flow cytometry, serum was analyzed for levels of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α as well as Th(1) cytokines IL1-b, IL-2, IL-12, IFN-g and Th(2) cytokines IL-4, IL-5, and IL-10.

Results: Both post transplant cohorts (Group 2 and 3) had higher mean levels of GM-CSF (110 and 120 pg/ml), IFN-g (17 and 16 pg/ml), and IL-12 (318 and 202 pg/ml) compared to their respective non-transplant (Group-1) controls (GM-CSF 23 pg/ml: p<.001; IFN-g 4 pg/ml: p<0.01; IL-12 60 pg/ml: p<0.05). Compared to group 2 patients, Group 3 patients with CT had significantly lower mean levels of TNF-α (46 vs 26pg/ml: p<0.03), IL-12 (318 vs 202 pg/ml: P<0.05) and IL-8 (83 vs 59: p<0.05). In contrast mean serum levels of IL-4 (43 vs 26 pg/ml: p<0.001) and IL-5 (51 vs 9 pg/ml: p<0.05) were significantly higher in group 3 CT patients compared to Group 2 patients without thrombocytopenia. When subdivided into groups based on the presence or absence of chronic GVHD, mean IL-4 levels were significantly higher in CT patients with or without CGVHD (33 and 44 pg/ml respectively) compared to post transplant patients without CGVHD with normal platelet counts (18pg/ml: p<0.05). In contrast, patients with CT with or without CGVHD had significantly lower mean levels of IL-8 (89 and 49 pg/ml respectively) compared to patients with or without CGVHD (200 and 126 pg/ml respectively: p<0.05) who did not have thrombocytopenia. We conclude that complex alterations in both pro and anti-inflammatory cytokines are involved in the pathophysiology of CT following allogeneic HCT; based on these data, studies investigating the therapeutic potential of IL-4 inhibition in patients developing CT might be considered.
Latent-Membrane 2a Specific CTL’s but Not Latent-Mebrane 1 Specific CTL’s Lysis Efficiently Hodgkin Lymphoma Cells.
Authors: Topp Max S. , Diekamm Jan , Beck Olaf , Rauser Georg , Hauschulz Kai , Neff Christina , Kuntz Gabriele , Einsele Hermann
Published: October 2019

Relapse is the leading cause of treatment failure after allogeneic SCT of Hodgkin Disease (HD). As Ebstein-Barr infection (EBV) is associated with 60% of all HD cases, adoptive immunotherapy with donor derived EBV-specific T-cells lines has resulted in disease control of allogeneic SCT. Potential targets for the adoptively transferred T-cells are the type II latency protein LMP-1 and LMP-2a, which are both homogenously expressed by HD cells. In healthy individuals, both LMP-1 and LMP-2a elicits subdominant CD8+ T-cell responses with frequencies of less than 1:10000. LMP-1 and LMP-2a specific T-cells from 1x108 PBMC derived from HLA A*0201+healthy donors were stimulated with the HLA A*0201 LMP1-epitopes YLLEMLWRL and YLGQNWWTL and the HLA A*0201 LMP-2a epitope CLGGLLTM. Activated T-cells were selected by the cytokine secretion assay and expanded for 10 days. In 85% of donors 1.7 x106 (range 0.7 –4.5 x106; n=13) LMP-1 or LMP-2a specific CD8+ T-cell could be generated with an average purity of 83% as determined by tetramer staining. LMP1- and LMP2a-specific CD8+ T-cells were then expanded 3000 x in 14 d by the rapid expansion protocol and evaluated functionally for cytokine production and specific lysis. Both LMP-1 and LMP-2a specific CD8+ T-cells retained specific cytokine production if stimulated with peptide pulsed targets, efficiently lysed peptid pulsed targets. Surprisingly, if LMP-1 was presented endogenously by EBV positive targets or by targets cells transduced with LMP-1, no cytokine production or specific lysis was detected despite protein expression of LMP-1 in all targets. In contrast, IFN-γ production could be readily detected in LMP-2a-specific CD8+ T-cells after stimulation with target cells processing endogenously the LMP-2a antigen as well as specific lysis of EBV positive target cells. Furthermore, LMP2a specific CD8+ demostrated also specific lyse of Hodgkin-cells expressing the LMP2a (30:1 E/T ratio; 29,3%) where as LMP-1-specific CD8+ T-cells could not lyse HD-cells. In summary, LMP-1 and LMP-2a specific T-cells, although present at undectable levels in healthy donors, can be readily selected and expanded to up to 6x109 antigen-specific T-cells in less than 4 weeks starting from 1x108 PBMC. Based on this data, adoptive immunotherapy of relapsed EBV positive HD after allogeneic SCT should be preferentially performed with LMP-2a specific CD8+ T-cells rather than with LMP-1 specific CD8+ T-cells.
An Interim Report Experience on Acute Leukemia and High Dose Therapy in Jehovah’s Witnesses Treated in a Single Institution.
Authors: Mazza Patrizio P. , Palazzo Giulia G. , Amurri Barbara B. , Pisapia Giovanni G. , Pricolo Giancarla G. , Prudenzano Antonella A. , Stani Loredana L.
Published: October 2019

The major difficulty in the treatment of Jehovah’s Witnesses with haematological malignancies is the deny to the transfusion of blood products as support to chemo-radiotherapy; since the use of any form of bone marrow transplantation it is instead not precluded, this may be an alternative. From 1997 to July 2004 we treated 9 Jehovah’s Witnesses with de novo acute leukaemia without transfusions of blood products; in the same period 10 Jehovah’s Witnesses with various malignancies received high-dose therapy as conditioning to autologous PBSCT or allogeneic BMT. Acute leukaemia consisted of FAB-M3 4 patients (pts), FAB-M4 1 pt, FAB-L1 1 pt, FAB-L2 2 pts, FAB-L3 1 pt. Hb at diagnosis was 10.3g/dL mean (5.5–14.0 range), PLT at diagnosis were 40x10^3/μL mean (5–197 range). The mean age of pts was 26.2 years (2–49 range). The treatment consisted of protocols in use in our Institution at the time of diagnosis. The nadir of Hb was 5.3g/dL mean (1.3–8.3 range) and it was reached 10 days mean from start of therapy (1–16 days range); the nadir of PLT was 16x10^3/μL mean (0–70 range) and was reached 6 days mean from start of therapy (1–13 days range). The results to therapy show that 8/9 pts reached a CR and 1 died for anaemia during induction when the Hb was 1.3g/dL; at the follow-up 2 pts relapsed 3 and 8 months, respectively and died for disease progression, 6 pts are alive and in CR 12–60 months following the end of therapy. The patients undergoing high-dose therapy included NHL 1 pt, ALL 2 pts, CLL 1 pt, Breast Cancer 1 pt, CML chronic phase 1 pt, CML blastic phase 1 pt, MDS 1 pt, MM 1 pt and AML 1 pt; 6 of them received autologous PBSCT and 4 allogeneic BMT. Three pts were in CR following first-line therapy, 5 in relapse and 2 with progressive untreated disease. High-dose therapy consisted of classic Thiotepa and Melphalan, CTX and TBI, BEAM and Melphalan alone depending the disease. Hb before high-dose therapy was 12.2g/dL mean (6.2–14.0 range) and PLT were 186x10^3/μL mean (10–328 range). The nadir of Hb was 7.2g/dL mean (2.0–11.0 range) and that of PLT 6.0x10^3/μL (1–14 range). The recovery of reticulocytes (>20x10^3/μL) was reached at 12 days from HDT mean (7–22 range) and PLT (>20x10^3/μL) at 13 days mean (8–27 range). None of patients occurred major bleeding or complications due to anaemia. One pt died for acute GVHD 2 months following TMO, 3 pts died for disease progression and 6 pts are alive in CR 2–48 months following HDT. In conclusion this study demonstrates that therapy for acute leukaemia and high-dose therapy as conditioning to bone marrow transplant are feasible in patients refusing transfusions of blood products and the results are almost similar to comparable patients accepting blood transfusions.
The Effects of Super-Intensified Conditioning Regimen of Allogeneic Hematopoietic Stem Cell Transplantation for Refractory Leukemia.
Authors: Liu Qifa , Sun Jing , Xu Dan , Zhang Yu , Fan Zhiping , Wei Yongqiang , Meng Fan Yi , Zhou Shuyun
Published: October 2019

Objective To explore the regimen related toxicity (RRT) and the effects of super-intensified conditioning regimen combined with inducing graft versus leukemia (GVL) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for refractory leukemia which can’t obtain complete remission (CR) pre- transplantation.

Methods 18 patients who did not obtain CR before transplantation received super-intensity conditioning regimen protocol(experimental group), and 62 patients with acute leukemia who obtained CR or chronic myeloid leukemian (CML) who were in the chronic phase before transplantation received total body irradiation (TBI) plus cyclophosphamide (CTX) or modified BuCY (hydroxyurea, busulfan, Ara-C, CTX) protocol (control group). Cyclosporin A was reduced rapidly and gradually or donor lymphocytic infusion (DLI) was used to induced GVL if acute graft versus host disease (GVHD) did not happened in patients with refractory leukemia at 30 days post-transplantation. The incidence and mortality of RRT during transplantation, and the rate of CR, GVHD and leukemia relapse after transplantation was investigated. Kaplan-Meier survival analysis model was used to estimate the disease-free survival (DSF) rate at 3 years post-transplantation.

Results Except one patient in experimental group and two patients in control group died of transplant-related complications, all the other patients obtained hematopoietic reconstitution.’ the total RRT incidence were both 100% in two groups. The RRT of stomach intestine were most common in all the organs and the RRT incidence of experimental group and control group was 83.3% and 85.5%, respectively, in stomach intestine. The RRT incidence was 44.4% and 62.9% in oral cavity and 16.7% and 33.9% in bladder, respectively, in the experimental group and control group. There was no significance and P value was 0.823, 0.172 and 0.244, respectively, in the RRT incidence of stomach intestine, oral cavity and bladder between the two groups. The RRT mortality was 0 and 5%, respectively, and was not different (P=0.341) in the experimental and control group. Except one patient died of infection, all the other patients obtained CR in patients who were treated with supper-intensified conditioning regimen. The incidences of acute or chronic GVHD were 58.8% and 40.0% or 92.6% and 55.8%, respectively, in the experimental and control group. The incidence of leukemia relapse was 11.8% and 18.3%, respectively, in the two groups. The DSF at 3 years after transplantation was 61.2±12.3% and 65.0±7.4% (P=0.6311), respectively, in the two groups.

Conclusion The consecutive super-intensified conditioning regimen combined with inducing GVL post transplantation protocol can increase the rate of CR and DFS, and dose not increase RRT incidence and mortality in allo-HSCT for the refractory leukemia which can’t obtain CR pre- transplantation.