bluuebunny/crossref_metadata_2025 huggingface dataset:
PT and INR Interference by the Antibiotic Daptomycin in Commercial Thromboplastin Reagents: Dependence on Phospholipid Composition.
Authors: Cawthern Kevin , Bottenus Ralph , Rawal Dharini , Triscott Mark
Published: October 2019

The antibiotic, daptomycin (brand name Cubicin, Cubist Pharmaceuticals) is approved in the US for treatment of complicated skin and skin structure infections caused by gram-positive pathogens (e.g., MRSA, methicillin-resistant Staphylococcus aureus). Recently, this antibiotic has been shown to interfere with prothrombin time (PT) assays, resulting in a prolongation of clotting times and artificial increases in the INR (International Normalized Ratio). The level of interference varies with Daptomycin concentration in the plasma, the brand of prothrombin time reagent as well as the type (normal or abnormal) of patient sample being analyzed. The bactericidal action of the antibiotic is known and is through a depolarization of bacterial membranes in the presence of physiologic calcium (Silverman, et al, Antimicrobial Agents and Chemotherapy, Aug 2003); therefore, daptomycin interference with the PT clotting assay is likely mediated through the phospholipid component of the thromboplastin formulations. To examine the dependence of daptomycin interference for a number of commercial PT formulations, testing was performed with daptomycin spiked samples of normal pooled plasma as well as abnormal samples from oral anticoagulant therapy (OAT) patients. For spiked normal samples, a small degree of prolongation in clotting times was observed with most PT reagents, with one reagent showing a significant prolongation. In OAT samples spiked with Daptomycin, the PT prolongation was greater for all formulations, with the one reagent again showing greater prolongation as for the normals. Ultimately, this variation in response was attributed to differences in phospholipid composition among the reagents, supported by previous studies of daptomycin with neutral and acidic phospholipids in the presence of calcium (D. Jung, et al., Chemistry and Biology, Jul 2004). A four-dimensional experimental design matrix was constructed using a mixture of four synthetic phospholipids (DOPC, DOPS, DOPE and DOPG), and the relationship of daptomycin-PT interference to percent composition of the phospholipids was examined. In both normal and OAT plasmas spiked with daptomycin, the major contributor to PT prolongation was the concentration of DOPG (phosphatidylglycerol) in the lipid mixture, with additional contributions correlated with DOPC (phosphatidylcholine). Because of the ubiquitous use of these phospholipids in commercial PT formulations, this work suggests that daptomycin interference should be assessed for each combination of PT reagent and INR range used.
Peroxisome Proliferator-Activated Receptor gamma (PPARγ) Regulates CD36 Gene Expression in Megakaryocytic Cell Lines.
Authors: Thornton Michael A. , Poncz Mortimer , Badejogbin Victor
Published: October 2019

CD36 is a receptor for thrombospondin and is present on megakaryocytes and platelets. It may be critical for normal megakaryocyte-formation and platelet function. The human CD36 gene locus extends 38.3 kb along chromosome 7 at position 7q11.2. Six distinct promoters (1A-1F) have been described that flank corresponding alternatively spliced first exons. To date the activity of these six promoters has not been tested in the megakaryocytic lineage and understanding the regulated expression of the CD36 gene may provide additional insights into the biology of this receptor. The 1B promoter, which is active in monocytes and adipocytes possesses a PPAR γ Direct Repeat (DR-1) binding site at position -260bp that is inducible in those same lineages with PPAR γ ligands. Recent studies have shown that primary megakaryocytes, platelets and the megakaryocyte line MEG-01 cells express PPARγ. To elucidate the molecular basis for CD36 gene expression in megakaryocytes and to query whether PPARγ plays a role in CD36 expression in this lineage we treated two megakaryocytic cell lines HEL and CHRF with increasing doses of two PPARγ ligands, 15DPGJ2, an arachadonic acid (AA) metabolite and Troglitazone, a thiazolidinedione drug administered to diabetics. RT-PCR studies using an exon 1A/exon 3 primer set or an exon 1B/exon 3 primer set established that CD36 mRNA from both promoters was expressed in these two lineages. Further these studies showed that HEL cells exhibited ligand dependent transactivation of the 1B but not the 1A promoter to both 15DPGJ2 and Troglitazone. CHRF cells did not demonstrate a drug dependent induction of either promoter. However, flow cytometry studies using a FITC conjugated anti-human CD36 antibody revealed that both HEL and CHRF cells had significant increased surface expression of the CD36 receptor. CHRF cells in particular had a 23 fold increase in surface CD36 over vehicle treated controls. Western Blot Analysis demonstrated that this increase in surface receptor quantity was secondary to drug-induced increases in the steady state protein levels. The relative increase in protein level was consistent with the mRNA levels determined by our RT-PCR results. We conclude that the 1B promoter is PPARγ responsive in HEL cells and that an alternate promoter perhaps 1C, 1D, 1E, 1F or a novel promoter is PPAR gamma responsive in CHRF cells. These data may have clinical implications for diabetics currently undergoing thiazolidinedione drug treatment with troglitazone as CD36 increases on the platelet surface lead to increased AA uptake and circulating platelet hyperactivation. These represent the first studies showing the nuclear receptor PPARγ’s involvement in regulating gene expression in megakaryocytes.

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Human Mesenchymal Stem Cells Express HLA-G: Role in MSC Mediated Immunosuppressive Effect.
Authors: Chapel Alain , Nasef Aisha , Mathieu Noelle , Frick Johanna , Bouchet Sandrine , Gorin Norbert C. , Thierry Dominique , Fouillard Loic
Published: October 2019

Mesenchymal stem cells (MSC) possess immunomodulatory properties, being able to suppress allogenic T cell response and modify maturation of antigen presenting cells. Recently its role for the treatment of graft versus host disease has been reported. The mechanisms of immunosuppression induced by MSC are being currently investigated.

HLA-G is a non-classical major histocompatibility complex (MHC) class I antigen HLA-G known by its strong immune-inhibtory property. We examined if HLA-G plays a role as a mediator of MSC induced immunosuppression. The expression of HLA-G on human MSC alone and in mixed lymphocytes reaction MSC/MLR was analyzed.

We found that HLA-G can be detected by RT-PCR, immunofluorescence, and by flow cytometry using intracellular staining (52.4 % ± 3.6). Using ELISA a level of 38.7 ± 5.2 ng/ml of soluble HLA-G was detected in the culture supernatant. HLA-G protein expression level is constitutive and is not modified upon stimulation by allogenic lymphocytes in MSC/MLR. We analysed the functional role of HLA-G protein expressed by MSC using anti HLA-G blocking antibody (87G). We found that blocking HLA-G protein can restore lymphocytes proliferation in MSC/MLR (25.4%, P= 0.04).

Our findings provide evidence supporting the immunosuppressive properties of MSC and the role of HLA-G. It emphasizes the potential application of MSC as a relevant therapeutic candidate in transplantation.
A Novel Translocation (4;12)(q11;q13) in an Acute Promyelocytic Leukemia-Like Case Lacking RARα Rearrangements.
Authors: Qiu Hui Y. , Xue Yong Q. , Pan Jin L. , Wu Ya F. , Wang Yong , Shen Juan , Zhang Jun
Published: October 2019

We report a novel chromosomal translocation t(4;12)(q11;q13) in a 37-year-old male with acute promyelocytic leukemia (APL)-like morphologic changes but lacking RARα rearrangements. His blast cells had some morphologic features evocative APL such as heavy azurophilic granules, bundles of Auer rods except regular round or oval nuclei. Immunophenotype of blast cells showed positivity for CD13 and CD33, and negativity for CD34 and HLA-DR which were compatible with the diagnosis of APL. Disseminated intravascular coagulation (DIC) was present. Chromosome study of BM cells showed that 29 out of 35 metaphases had a consistent karyotype of 46, XY, t (4;12)(q11;q13) which was confirmed by chromosome painting with whole chromosome paint probes 4 and 12. However, fluorescence in site hybridization, reverse transcriptase-polymerase chain reaction (RT-PCR) and multiplex RT-PCR did not demonstrate any evidence for RARα rearrangements including PML-RARα, PLZF-RARα, NuMA-RARα, NPM-RARαand STAT5b-RARα fusion genes. Sequential treatment with arsenic trioxide and chemotherapy had no effect, he died of bleeding due to DIC. In view of the fact as mentioned above, this case should be rated as transitional M2–M3.
Retroviral Insertional Mutagenesis Screen in a C/EBPalpha Proliferative Genetic Background.
Authors: Hasemann Marie S. , Sørensen Annette B. , Pedersen Finn S. , Nerlov Claus , Porse Bo
Published: October 2019

The CCAAT enhancer binding protein alpha (C/EBPalpha) transcription factor plays a key role in the regulation of growth and differentiation of the granulocytic lineage in the hematopoietic system. Consistently, mice lacking C/EBPalpha have no mature neutrophils and die within a few hours after birth. In contrast, homozygous knockin mice in which the wild type Cebpa allele has been replaced with a mutant allele (BRM2) deficient in repressing the activity of E2F family members, are viable. At 8 weeks of age these animals display myeloid dysplasia with absence of neutrophil granulocytes. Strikingly, in older BRM2/BRM2 knockin mice the myeloid dysplastic phenotype progress into other myeloid malignancies such as myeloid proliferative syndrome and acute myeloid leukemia. These findings strongly suggest that secondary mutations in other loci must occur during the phenotypic progression.

In order to identify genes that cooperate with C/EBPalpha in the development of leukemia in BRM2/BRM2 mice a so-called retroviral insertion mutagenesis screen was performed. Inbred newborn BRM2/BRM2 and wildtype mice were injected with SRS19-6 retrovirus and when disease is evident the mice are euthanized and analyzed. As expected the BRM2/BRM2 mice have a shorter latency than wildtype mice (182 vs. 260 days). The mice have enlarged spleen, thymus, and lymph nodes and were further characterized by histology, flow cytometry and Southern blotting in order to determine the hematopoietic phenotypes. Most abundantly was the AML-like phenotype, but also T-cell lymphomas are developing. Finally, the loci carrying retroviral insertions loci are identified through a splinkerette-aided PCR strategy.

This study provides a better understanding of the genes involved in the development of myeloid leukemia.
Deregulation of c-Myc in Primary Effusion Lymphoma by KSHV LANA.
Authors: Bubman Darya , Guasparri Ilaria , Cesarman Ethel
Published: October 2019

Primary effusion lymphoma (PEL) is a rare subtype of non-Hodgkin’s lymphoma, which is associated with infection by Kaposi’s sarcoma herpesvirus (KSHV/HHV-8). PELs lack expression of B-cell markers and rearrangements in common oncogenes. c-Myc is a transcription factor that plays an important role in cellular proliferation, differentiation and apoptosis. Lymphomas frequently have deregulated c-Myc expression due to chromosomal translocations, amplifications or abnormal stabilization. However, no structural abnormalities were found in the c-Myc oncogene in PELs. Given that c-Myc is often involved in lymphomagenesis, we hypothesized that it is deregulated in PELs. We report that PELs have abnormally stable c-Myc protein. Normally, the turnover of c-Myc protein is stringently regulated by post-transcriptional modifications, which include phosphorylation of c-Myc threonine 58 (T58) by glycogen synthase-3 beta (GSK-3 beta). Our data show that the impaired c-Myc degradation in PELs is associated with drastic underphosphorylation of c-Myc T58. The KSHV latency-associated nuclear antigen (LANA) is responsible for this deregulation. Overexpression of LANA in HEK 293 cells leads to abnormal stabilization of c-Myc protein and inhibition of GSK-3 beta-mediated phosphorylation of c-Myc T58. LANA also increases c-Myc protein, but not RNA levels when overexpressed in primary B cells. Conversely, when LANA is eliminated from PELs using RNA interference, there is a post-transcriptional effect leading to a reduction in c-Myc protein. The ability of LANA to prolong the half-life of c-Myc might play an important role in the pathobiology of PEL.
Expression of Multidrug Resistance 1, Lung Resistance Protein and Breast Cancer Resistance Protein Genes in Chronic Lymphocytic and Chronic Myelogeneous Leukemia.
Authors: Ural A. Ugur , Iseri Ozlem Darcansoy , Mutlu Pelin Kaya , Kars Meltem Demirel , Avcu Ferit , Gunduz Ufuk
Published: October 2019

One of the major problems in the treatment of leukemia is the development of drug resistance to chemotherapeutic agents, which is already present at diagnosis or after chemotherapy as a minimal residual disease. The resistance may be originated from genetic or epigenetic mutations during prior growth of the leukemic clone. In this study, the expressions of three multidrug resistance (MDR) genes were investigated. Expression levels of multidrug resistance resistance gene 1 (MDR1), breast cancer resistance protein (BCRP) gene, and lung resistance protein (LRP) gene expression levels were determined in peripheral blood samples from 16 cases of chronic lymphocytic leukemia (CLL) and from 23 cases of chronic myelogeneous leukemia (CML) using RT-PCR. The expression of each of these genes was then expressed as a ratio in relation to β2-microglobulin gene expression in densitometric measurements. MDR1, BCRP and LRP expression levels was detected in 56,3%, 18,8% and 50% of CLL patients, respectively, with no difference for stage or response to the treatment. Four out of 16 (25%) CLL patients expressed none of these genes. The other 25% CLL patients expressed all of these genes. MDR1, BCRP and LRP expression was detected in 47,8%, 39,1% and 30,4% of CML patients, respectively, with no difference for progression or response to imatinib mesylate. Eight out of 23 (34,8%) CML patients expressed none of these genes. Four out of 23 (17,4%) CML patients expressed all of these genes, and two out of whom rapidly progressed to acute leukemia and unresponsive to the treatment. In conclusion, MDR and LRP overexpression seems to be a frequent event in CLL and CML patients; however no conclusion can be drawn on their prognostic role and response to the treatment.
Genetic Mechanisms of Cytarabine Resistance in Leukemia: Cell Culture and Mouse Transplantation Models.
Authors: Yin Bin , Diers Miechaleen D. , Rathe Susan K. , Lowe Scott W. , Largaespada David A.
Published: October 2019

Chemoresistance remains a major barrier to successful clinical treatment of acute myeloid leukemia (AML). However, the underlying molecular mechanisms of chemoresistance in AML are largely unknown. Previously, we reported that loss of tumor suppressor genes, Nf1 and Trp53, can confer upon myeloid leukemia cells partial resistance to cytarabine (Ara-C), a mainstay of AML chemotherapy. Microarray gene expression profiling revealed that a group of lysosome function-related genes, and a major regulator of apoptosis, Bcl-xL, were upregulated in in vitro Ara-C resistant myeloid leukemia cell lines. We now report that the lysosome number and size is increased in resistant cells. Also, overexpression of Bcl-xL in both murine and human myeloid leukemia cell lines increased Ara-C resistance to a variant extent in different lines. Furthermore, we found that the key enzyme involved in intracellular activation of Ara-C, Cda, can do so, too, but to a less extent than Bcl-xL. Interestingly, in our Ara-C resistant AML cell lines which were retrovirally induced in BXH-2 stain of mice, we were able to detected new proviral insertion sites which may cause mutations associated with the leukemia resistance feature. We have now examined the possibility of modeling AML chemoresistance in mouse, and through this model, to identify and characterize AML relapse-related genetic abnormalities. During establishing such a model, treatment dosage, intervals, courses, routes of administration, the number of injection, food preparation and supplements have been considered for testing. Ara-C treatment caused a decrease in body weights and peripheral blood cell counts. One major lethal toxicity response, intestine deterioration, appeared to be correlated with high doses of Ara-C. We arrived at a regimen comprised of two 10-day treatment courses at 90 mg/kg/day by one intraperitoneal injection, with a 5-day break between treatment courses. This treatment course was found to give rise to 10~30% lethality due to Ara-C toxicity but it can also suppress the onset of transplanted AML disease in recipient mice for nearly two weeks compared to saline-treated mice as a control. Interestingly, the tumors collected from Ara-C treated transplants displayed higher resistance than saline treated tumors in a methylcellulose colony-forming assay. Testing of apoptosis regulatory genes for Ara-C response in primary tumors is in process.
Antiproliferative and Proapoptotic Effects of Akacid-Medical-Formulation on Hematologic Cell Lines.
Authors: Neuwirt Hannes , Salvador Christina , Wabnig Elisabeth , Tiefenthaler Martin , Culig Zoran , Konwalinka Guenther
Published: October 2019

We have previously shown that Akacid-medical-formulation (AMF) exerts its antiproliferative effects on various malignant solid cell lines, including those derived from prostate via downregulation of mitogen-activated protein kinases Erk 1/2 and cell cycle regulators, e.g. cdk-2, -4, cyclin E, -D1 (Neuwirt et al. 2006). However, the effect of AMF on proliferation and induction of apoptosis in malignant hematologic cell lines has not been investigated yet. Therefore, we performed 3H-thymidine incorporation assays to assess the growth inhibition of HL-60, U-937, K-562, CEM-C7H2 cells after treatment for 48 hours with various concentrations of AMF (0.3 – 100 μM). All cell lines were dose-dependently inhibited by AMF with an IC50 of about 2.1μM. As cellular growth arrest is known to be one major reason for resistance to chemotherapeutics, we investigated the effect of AMF on the cell line CEM-C7H2-6E2, in which G1/0-phase arrest can be induced by tetracyclin-regulated expression of the cell cycle inhibitor p16INK4A (Fig. 1A). Flow cytometric analysis using Annexin-V / propidium-iodide staining showed that in G1-phase arrested cells apoptosis was induced to a similar extent (LD50 of 10μM) as compared to proliferating control cells (CEM-C7H2-2C8) (Fig. 1B). In addition, we tried to find out whether AMF induces apoptosis via the caspase-9 dependent intrinsic or the caspase-8 dependent extrinsic pathway. For this purpose, two different cell lines stably transfected with tetracyclin-responsible plasmids encoding for the antiapoptotic proteins bcl-2 (CEM-C7H2-10E1) and CrmA (CEM-C7H2-2E8) were used. After 48 hours of treatment with AMF (1 – 30μM) we found that overexpression of neither bcl-2 nor CrmA could inhibit AMF-induced apoptosis compared to control cells (LD50 15μM). Data on apoptosis obtained by flow cytometry were confirmed by Western blot analysis on activated caspase-8, -9, -3, and cleaved PARP in CEM-C7H2 cells. Caspase-8 and -9 were not activated after 48 hours. However, it is interesting that downstream effectors of apoptosis, cleaved PARP and caspase-3, were strongly induced (1000 % of control at 30μM). In conclusion, our results show a potent antiproliferative effect of AMF on leukemic cell lines. Induction of apoptosis could not be inhibited either by G1-phase arrest or overexpression of the antiapoptotic proteins bcl-2 or CrmA. Although we found a substantial activation of the downstream effectors of apoptosis by AMF including caspase-3 and PARP, the upstream pathway of activation remains unclear.

Figure 1 Figure 1.
Molecular Relapse Monitoring and Treatment in Acute Promyelocytic Leukemia (APL) Patients.
Authors: Shuravina E.N. , Parovitchnikova E.N. , Misyurin A.V. , Demidova I.A. , Isaev V.G. , Savchenko V.G.
Published: October 2019

Molecular monitoring of PML/RARA chimeric gene expression is stated to be necessary for diagnosis of acute promyelocytic leukemia (APL), as well as for monitoring of minimal residual disease (MRD). In our study we aimed to modify leukemia treatment according to MRD results. PML/RARA expression was measured by RT-PCR. Synthesis of primers and RT-PCR were performed using recommendations of BIOMED-1 Concerted Action (1999). Sensitivity of method was estimated as 10−4 – 10−6. Fresh bone marrow (BM) aspirates were investigated before treatment, during induction and consolidation, every 2–3 months during maintenance therapy, and after completion of treatment. A total of 73 morphologically suspected APL patients were included in the study; expression of PML/RARA was found in 69 patients (94.5%), bcr1/2 type in 31 patients (45%), and bcr3 type in 38 patients(55%). MRD screening during maintenance therapy was performed in 47 PML/RARA-positive APL patients. Median follow-up was 4 years (2 mo – 8 years). If chimerical transcript was revealed in a BM sample once, this was considered probable molecular relapse, and when PML/RARA was find twice in 2–3 weeks, we considered this proven molecular relapse. Molecular relapses were revealed in 19 of 47 (40.4 %) APL patients. In 6 of 19 patients (31.5 %) relapses were proven, and in 13 (68.5%) relapses were probable. When the molecular relapses were detected, maintenance chemotherapy was changed in 3 of 13 (23%) patients with probable relapses and in 5 of 6 (83%) patients with proven relapses. None of them developed hematological relapse. Therapy was not changed in 11 patients, and 3 of 10 (30%) patients with probable molecular relapse and 1 pt with proven relapse demonstrated recurrence of the disease. Thus, 4 of 11 (36%) patients with unchanged therapy developed hematological relapse. These results are significantly different from patients with modified maintenance treatment (P=0.01). Interesting data were obtained while investigating BM samples obtained from 5 patients in whom BM sampling were performed at 2 different sites (sternal and iliac). Simultaneously performed RT-PCR of those samples showed different results in 3 of 5 (60%) patients.

Though we have a small cohort of patients, we may conclude that it is advisable to change chemotherapy when proved or probable molecular relapse is revealed., When the single positive result of chimerical transcript expression is obtained, it is important to repeat RT-PCR analysis in 2–4 weeks in order to prove the molecular relapse, and then to adjust the maintenance program. However, even when it is impossible to perform the second analysis, changing the therapy may be advisable.
Detection of Chromosomal Aberrations in CLL and Correlation with Clinical Staging.
Authors: Durak Beyhan , Akay Meltem Olga , Kaytaz Behiye , Burul Ismigul , Ozdemir Muhsin , Artan Sevilhan , Gulbas Zafer
Published: October 2019

Chromosome abnormalities detected in CLL are important markers for determination of treatment approaches. This study was aimed to evaluate usefulness of FISH panels for testing chromosome aberrations in CLL and to correlate the aberrations with clinical staging of disease and responses to chlorombucil therapy. Cytogenetics and FISH analyses from bone marrow samples of 67 CLL patients were performed according to standard unstimulated short term culture methods. CLL specific FISH panel included probes for 11q22.3 (LSIATM), CEP12, 13q14 (LSID13S19 and LSID13S25), 17p13.1 (LSIp53). FISH analysis was performed in all samples. The most commonly seen abnormality was 13q14 deletion followed by deletions of ATM, p53 and trisomy12. Of 19 cases with 13q14 deletion, 11 had only this abnormality whereas the others had trisomy12, p53 and ATM loci deletions (multiple abnormalities). Of 11 cases with single abnormality, 8 were at stage A, 3 was at stage C and all patients achieved benefits from chlorombucil therapy except one patient. Failed therapy response was seen in 9 patients with multiple abnormalities and the disease was at stage C (4 cases) and B (5 cases). Patients showing p53 and ATM deletions related with poor prognosis had progressive disease and no benefit could be achieved from chlorombucil therapy. Of 6 cases with trisomy 12, 4 were at early stage and response to the therapy was good but in two cases at later stage, therapy benefit could be achieved in one while no benefit was seen in the other one. The results showed that FISH analysis is more informative than karyotype analysis in CLL. Detection of p53 and ATM loci deletions is an important marker for administration of second level therapy.
Myelodysplasia Following Therapy with Tumour Necrosis Factor-alpha (TNF α) Inhibitors - An Uncommon Occurence.
Authors: Kamat Anil V. , Ezekwesili Raphael , El-Miedany Yasser
Published: October 2019

Introduction-Tumour necrosis factor- alpha (TNF α) inhibitors such as infliximab & etanercept have various applications such as severe rheumatoid arthritis, seronegative spondyloarthritis, vasculitis, crohn’s disease & psoriasis. There have been few reports of haematological complications arising in patients treated with anti-TNF a therapies. There has been only one reported case of development of acute myeloid leukaemia after initiation of etanercept therapy.

Case report-A 57year old gentleman with HLA B27 negative ankylosing spondylitis presented with chest infection. His full blood count showed Hb 5.9 g/l WBC 3.8 × 10^9/L Neutrophil count 2.7 × 10^9/L Lymphocyte count 0.7 × 10^9/L Platelet count 89 × 10^9/L & Reticulocyte count 21 × 10^9/L. Occasional myeloblasts were seen on the blood film. He had been on biological therapy for ankylosing spondylitis with infliximab 5mg/kg infusion on 8 weekly basis for 18 months which was stopped due to lack of efficacy. Subsequently he had been treated with etanercept 50 mg subcutaneous once weekly for the past 16 weeks prior to this presentation. His full blood count prior to initiation of infliximab & etanercept was Hb 11.6 g/l WBC 16 × 10^9/L Neutrophil count 12.8 × 10^9/L Lymphocyte count 2.1 × 10^9/L Platelet count 655 × 10^9/L and Hb 11.0 g/l WBC 8.2 × 10^9/L Neutrophil count 4.9 × 10^9/L Lymphocyte count 2.4 × 10^9/L Platelet count 591 × 10^9/L, respectively. He underwent bone marrow investigations. Bone marrow aspirate was aparticulate with dyshaemopoeisis. Dyserythropoeisis in form of irregular nuclei, occasional binucleated cells & delayed nucleo-cytoplasmic maturation was seen along with dysmyelopoeisis in form of binucleated cells & pelger forms. Blasts amounted to 14% of nucleated cells. Overall, impression was in keeping with refractory anemia with excess of blasts (RAEB-2). Trephine biopsy showed mildly hypocellular bone marrow with markedly disordered haemopoeisis. There was trilineage dysplasia with abundant micromegakaryocytes with immunocytochemistry for CD117 showing increased number of progenitors (10–20%). The appearances were in keeping with myelodysplasia with excess of CD117 positive progenitors. Cytogenetic study failed. The sepsis responded to broad spectrum antibiotics but he continues on red cell & platelet support on a weekly basis.

Conclusion The chronology points to a TNF α inhibitor mediated effect in this patient. Despite the close temporal association between exposure to TNF α inhibitor therapy and the presentation of myelodysplasia in this patient, a causal relationship cannot be established with confidence. Tumour necrosis factor inhibitors find wide applications in inflammatory conditions with high TNF. Such treatment may be associated with uncommon complications. It is of interest to note there are ongoing trials using TNF α inhibitors in myelodysplastic syndrome. Checking a full blood count periodically, and immediately if the patient is unwell is recommended.
The Investigation of JAK2 V617F Mutation in Chinese Patients with Hematological Malignancies.
Authors: Zhang Sujiang , Li Jianyong , Li Weida , Song Junhong , Duan Limin , Lu Mize , Qiu Hongxia
Published: October 2019

Myeloproliferative Diseases (MPD) are a spectrum of pathogenetically related disorders of varying clinical manifestations, characterized by neoplastic expansion of relatively mature granulocyte, erythroid, megakaryocyte, or monocyte and eosinocyte lineage cells. Recently a novel point mutation affecting the Janus tyrosine kinase 2 (JAK2 V617F) was identified as pathogenetically mechanisms by multiple competing groups. To investigate its prevalence and clinical significance in Chinese patients with hematological malignancies, we introduced Allele-specific PCR (AS-PCR) combined with sequence analysis to screen JAK2 V617F mutation.

A total of 98 Chinese MPD patients and 120 additional hematological malignancies including AML, ALL, MDS were analyzed for the JAK2 V617F mutation. 98 MPD patients were referred for PV (n=57), ET (n=18), IMF (n=12), HES (n=2) and CML (n=9). 2 ml peripheral blood samples of MPD and 5–10 ml bone marrow samples of AML, ALL, MDS at the time of initial diagnosis were obtained with informed consent and genomic DNA was isolated presently. In addition, peripheral blood samples from 20 healthy donors were also collected as control. All samples were first screened by AS-PCR. The positive samples were subsequently confirmed by sequence analysis.

The results showed that JAK2 V617F mutation was detected in 43 of 57 PV patients (75.4%), 7 of 18 ET patients (38.9%) and 5 of 12 IMF patients (41.7%). None of the AML, ALL, MDS, CML was found JAK2 V617F. There is no statistical difference of JAK2 V617F positive ratio between PV, ET and IMF. Furthermore, the mutation was not detected in the HES patient and 20 healthy controls. There is no other mutations and polymorphisms throughout exon 12 of JAK2.

To our knowledge, this is the first report of JAK2 V617F mutation in a number of Chinese patients with hematological malignancies especially BCR/ABL-negative MPD. The incidence of JAK2 V617F of our study is a little lower compared with other publications especially in PV patients. The main reason may be firstly attributed to ethnic difference. In addition, with more MPD patients introduced and more sensitive methods such as ARMS-PCR or Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) applied, more JAK2 V617F mutation will be identified.
Gene Expression Signatures Separate B-Cell Chronic Lymphocytic Leukemia Prognostic Subgroups Defined by ZAP-70 and CD38 Expression Status.
Authors: Dürig Jan , Hüttmann Andreas , Klein-Hitpass Ludger , Sellmann Ludger , Dührsen Ulrich
Published: October 2019

B-cell chronic lymphocytic leukemia (B-CLL) is a heterogenous disease with a highly variable clinical course and analysis of ZAP-70 and CD38 expression on B-CLL cells allowed for identification of patients with good (ZAP-70−CD38−) and poor (ZAP-70+CD38+) prognosis. DNA microarray technology was employed to compare eight ZAP-70+CD38+ with eight ZAP-70−CD38− B-CLL cases. The expression of 358 genes differed significantly between the two subgroups including genes involved in B cell receptor signaling, angiogenesis and lymphomagenesis. Three of these genes, i.e. IRTA4/FcRH2, angiopoietin 2 and Pim2 were selected for further validating studies in a cohort of 94 B-CLL patients. IRTA4/FcRH2 expression as detected by flow cytometry was significantly lower in the poor prognosis subgroup as compared to ZAP-70−CD38− B-CLL cells. In healthy individuals IRTA4/FcRH2 protein expression was associated with a CD19+CD27+ memory cell phenotype. Angiopoietin 2 plasma concentrations were two-fold higher in the poor prognosis subgroup (p<0.05). Pim2 was significantly overexpressed in poor prognosis cases and Binet stage C. Disease progression may be related to proangiogenic processes and strong Pim2 expression.
Increased Expression Level of Noxa in Peripheral Versus Lymph Node Chronic Lymphocytic Leukemia Cells Is Correlated with Survival Capacity.
Authors: Hallaert Delfine Y.H. , Smit Laura A. , Spijker Rene , de Goeij Bart , Jaspers Annelieke , Kater Arnon P. , van Oers Marinus H. , van Noesel Carel J.M. , Eldering Eric
Published: October 2019

Background: The gradual accumulation of chronic lymphocytic leukemia (B-CLL) cells is presumed to derive from proliferation centers in lymph nodes and bone marrow. To what extent these cells possess the purported anti-apoptotic phenotype of peripheral B-CLL cells is unknown. Recently, we have described that in B-CLL samples from peripheral blood, aberrant apoptosis gene expression was not limited to protective changes but also included increased levels of pro-apoptotic BH3-only member Noxa. The functional consequence of this finding is not known, nor whether this aberrant apoptosis gene profile is also present in CLL proliferation centers.

Aim: To perform a functional comparison of apoptosis gene profiles from peripheral blood (PB) B-CLL versus lymph node (LN) proliferation centers.

Methods: Immunofluorescence microscopy, RT-Multiplex-Ligation-dependent Probe Amplification (RT-MLPA), Western Blot, Transduction, RNA interference.

Results: PB samples (>90% CD5/CD19/CD23+ B-CLL cells) from 16 B-CLL patients and LN samples from 9 B-CLL patients were included in this study. LN samples contained over 90% CD5/CD19/CD23+ lymphocytes with Ki67+ cells either scattered throughout the LN or in follicle-like structures. RNA samples were subjected to the RT-MLPA procedure which monitors expression of 34 apoptosis genes. Apart from expected differences in survivin and Bcl-xL, the most prominent distinction with PB B-CLL cells was the generally low levels of Noxa in LN samples.

Figure 1: Profiling of apoptosis genes in peripheral blood and lymph node samples of B-CLL patients Figure 1:. Profiling of apoptosis genes in peripheral blood and lymph node samples of B-CLL patients

A reduction in Noxa RNA and protein levels could also be obtained by in vitro stimulation of PB B-CLL with CD40. Direct manipulation of Noxa protein levels was achieved by proteasome inhibition in B-CLL cells and via RNAi in model cell lines. In all these instances the viability of the cells was inversely correlated with Noxa levels.

Conclusions: These data indicate that spontaneous apoptosis of PB B-CLL cells in vitro is linked with high Noxa levels. We propose that suppression of Noxa in the LN contributes to the persistence of B-CLL, and that therapeutic targeting of Noxa might be beneficial.
Myeloma Cell Proliferation Is Inhibitied by the Activation of Adenosine Monophosphate Activated Protein Kinase (AMPK).
Authors: Baumann Philipp , Mandl-Weber Sonja , Emmerich Bertold , Straka Christian , Franke Daniel , Schmidmaier Ralf
Published: October 2019

In multiple myeloma (MM), a network of cytokines in the bone marrow microenvironment promotes myeloma cell proliferation. Consequent inhibition of intracellular signalling in the myeloma cells seems to be a promising strategy to encounter disease progression.

The multiple myeloma cell lines U266, OPM-2, RPMI-8226 and NCI-H929 were incubated with the AMPK activators AICAr and D942. Basal and cytokine stimulated proliferation rates of myeloma cells were measured by the WST-1 assay. Alterations of the cell cycle were determined by flow cytometry after staining with propidium iodide. Intracellular signalling was shown by western blotting.

The AMPK activators 5-aminoimidazole-4-carboxamide (AICAr) and D942 induced inhibition of proliferation in multiple myeloma cell lines. AICAr also induced a S-phase cell cycle arrest in all four tested cell lines and led to phosphorylation and herewith activation of AMPK. Furthermore, the inhibition of a nucleoside transporter by nitrobenzyl-thio-9-β-D-ribofuranosylpurine (NBTI), inhibition of the adenosine kinase by iodotubericidine and inhibition of AMPK by AMPKI Compound C reversed AICAr effects, indicating that the cellular effects of AICAr were mediated by AMPK. Activation of AMPK inhibited basal extracellular-signal regulated kinase (ERK), mTOR and P70S6 kinase (P70S6K) signalling and blocked cytokine induced increase of proliferation, which again was due to inhibition of ERK and P70S6K signalling. Troglitazone, a representative of a group of anti-diabetic drugs, similarly inhibited myeloma cell proliferation, activated AMPK and decreased ERK and P70S6K signalling.

We demonstrate for the first time that myeloma cell proliferation is controlled by AMPK activity. Consequently, targeting this pathway by inhibitors like glitazones provides a novel strategy in myeloma therapy.
In Vivo Phycocyanin Treatment of S180 Bearing-Mice: Increase in Immunic Function and Inhibition of Tumor Growth.
Authors: Pan Ling , Hao Wei , Zhang Xuejun , Niu Zhiyun , Wang Fuxu , Dong Zuoren
Published: October 2019

Objectives: To explore the effect of Phycocyanin (PC), with or without Cyclophosphamide(CY), on the growth inhibition of S180 cells and on the immunity improvement of S180-burdened mice. And to explore the anti-tumor mechanism of Phycocyanin.

Methods: Each 0.2ml of S180 cells, a sarcoma cell line cells, at the concentration of 2×107 cells/ml, were inoculated into the right axilla of each test mouse. These mice were randomizely divided into 4 groups: PC-treated group (including 50−, 75− and 100mg/kg·d- subgroups); CY-treated group (20mg/kg·d); PC plus CY-treated group and normal saline(NS)-treated group which served as control group. 24 hours after the inoculation, PC or CY or PC plus CY or NS, at a final volume of 0.2ml, were injected to the abdominal cavity of each mouse in different group, and consecutively for 7 days. These mice were killed after the seventh treatment, then the peripheral blood, the whole tumor and the spleen were collected from each mouse. Each mouse’s tumor weight was measured and blood routine exam was done. The serum level of IL-2, IFN-γ and TNF-α in each mouse was measured with ELISA method and the result was shown as pg/ml. Spleen cells from each mouse were collected and cultured respectively with ConA for 36 hours, and splenic T lymphocyte proliferation was measured.

Results: PC inhibited the growth of S180 cells at all three tested dosages, 50mg/kg·d, 75mg/kg·d and 100mg/kg·d, and the inhibition rates were 41.41%, 37.37% and 36.36%, respectively. PC-treatment increased WBC counts in PB of tumor-bearing mice. The results also showed that PC-treatment and tumor-bearing situation increased the spleen weight and enhanced the splenic T lymphocyte proliferation induced by ConA. PC-treatment did not reduce the body weight of tumor-bearing mice. Among these three dosage, 50mg/kg·d was shown to have the best effect in the above mentioned parameters, as well as in the increase of IL-2, IFN-γ and TNF-α secretion. PC at dosages of 75mg/kg·d and 100mg/kg·d just increased the serum level of IFN-γ and TNF-α, without the increase of IL-2. Compared with PC-treatment, CY-treatment diminished serum level of IL-2, IFN-γ and TNF-α. PC (75mg/kg·d) combined with CY-treatment increased the tumor inhibition rate to 92.93%.

Conclusions: 1, PC-treatment inhibited the growth of S180 cells. PC plus CY treatment showed more potential effect of growth inhibition. 2, PC enhanced markedly the immune function of S180-bearing mice in terms of increasing the spleen weight and the serum level of IFN-γ and TNF-α, and enhancing T lymphocyte proliferation induced by ConA. Those might account for the anti-tumor mechanism of PC. 3, PC-treatment antagonized the decrease in WBC count induced by CY-treatment. No obvious side effect of PC was found in the present study. Therefore, PC alone or combine with other chemotherapy drugs may offer a new approach in the treatment of hematological malignancies.
Pre-Emptive Treatment with Cidofovir for Cytomegalovirus Antigenemia in Autologous Bone Marrow Recipient and CLL Patients on Therapy with Alemtuzumab.
Authors: Iannitto Emilio , Minardi Viviana , Mulè Antonino , Scalone Renato , Calvaruso Giuseppina , Crescimanno Alessandra , Biundo Concetta Lo , Liardo Eliana , Abbadessa Vincenzo , Musso Maurizio , Arista Serenella
Published: October 2019

Cytomegalovirus (CMV) is an important cause of morbidity and mortality in patients who have undergone severe immunosuppressive therapy. Ganciclovir continues to be the first choice for pre-emptive therapy, but it needs multiple intravenous daily administration for three weeks and may cause myelosuppression. Cidofovir is a non myelotoxic nucleotide analogue effective against CMV; its favourable pharmacokinetic profile allows a once-a-week dosing. We reviewed a database on 110 consecutive Autologous Stem Cell Transplant (ASCT) and that of 15 Chronic Lymphocytic Leukemia (CLL) patients treated with alemtuzumab. All patients were virologically monitored by quantification of pp65 antigenemia in peripheral blood. Cytomegalovirus infections were identified respectively, in 13 of 110 (12%) ASCT group and in 10 of 15 (66%) CLL group. Nine out 23 CMV reactivation showed manifestation of the infection. All patients were treated on outpatient basis.

Patients with a positive pp65 assay were treated with cidofovir 5 mg/kg once-a-week for two weeks followed by one or two doses every two weeks. Twenty-three patients (13 autologus, 10 alemtuzumab) had 23 episodes of CMV-pp65 detection treated with cidofovir. The first positive antigenemia occurred after a median of 36 days from starting treatment (range 5–20) and the median antigenemia level at first appearance was 2 (range 1– 89).

The treatment produced regression of symptoms in all cases and clearance of the virus in 21 (11 post-transplant 84%; 10 post alemtuzumab 100%), stained by CMV antigenemia. Median duration of therapy was 21 days (range 14–30 days) and the time to the first undetectable antigenemia was seven days (range7–28). We did not observe any further CMV reactivations, also in six of the ten patients who restarted treatment with alemtuzumab after the end of pre-emptive therapy. We did not observe any of the side effects potentially related to cidofovir administration: notably, none of the patients experienced renal toxicity, proteinuria, nausea or vomiting, ophthalmological or neurological toxicity. In our experience, pre-emptive therapy of CMV infection with cidofovir is safe and effective. In our opinion it could be considered an interesting alternative to ganciclovir for pre emptive therapy, particularly advantageous for treatment of CLL and ASCT ambulatory patients at low risk of developing CMV disease.
Does Hypergammaglobulinemia Predispose to Secondary Solid Tumors in Patients with Chronic Lymphocytic Leukemia?.
Authors: Patel Vijay , Singh Jaswinder , Malani Ashok , Skikne Barry , Haung Chao , Vanveldhuizen Peter , Kambhampati Suman
Published: October 2019

Background:Occurrence of second hematological malignancies in patients with chronic lymphocytic leukemia (CLL) is well known but development of secondary solid tumors has not been well documented. Isolated case reports indicate, CLL patients may have a predisposition to develop solid tumors. There is not enough literature available to support this hypothesis. We here intend to study the incidence of secondary solid tumors in CLL patients with regard to their treatment and immunoglobulin levels.

Methods: We reviewed the medical records of 323 CLL patients over the last 20 years at Veteran Affairs Medical Center, Kansas City. Broadly the patients were divided into two groups; the group who received chemotherapy for CLL(74/323) versus the group who did not receive treatment (249/323) and their median immunoglobulin levels were also documented. Patients who developed secondary hematological malignancies were excluded from this study.

Results: The overall incidence of solid tumors was found to be 14.8% (48/323). In the chemotherapy treated CLL, the incidence of solid tumors was 18%(14/74) as compared to 13.6% (34/249) in the non-therapeutic group(P value 0.12). The most common malignancies noted were gastro-intestinal malignancies, genitor-urinary tumors and skin cancers including melanomas. Interestingly CLL patients who developed secondary solid tumors had a higher median IgG levels (1010 g/L) when compared to those who did not develop secondary tumors (738 g/L, P value <0.003)

Conclusion: Our study shows that there is a higher overall incidence of secondary solid tumors in patients with CLL when compared with incidence in the general population. There is no significant increase in the incidence of therapy related solid tumors in CLL patients. Upward drift in immunoglobulin levels should raise a suspicion for diagnosis of secondary solid tumors in patients with CLL. More studies are warranted to confirm this finding.
Reduced-Intensity Cord Blood Transplantation in Adult Patients for the Recurrence after Allogeneic Transplantation.
Authors: Seo Sachiko , Uchida Naoyuki , Yamamoto Hisashi , Matsuno Naofumi , Matsuhashi Yoshiko , Takagi Shinsuke , Kato Daisuke , Masuoka Kazuhiro , Wake Atsushi , Miyakoshi Shigesaburo , Taniguchi Shuichi
Published: October 2019

Backgrounds: For more than 10 years, umbilical cord blood has become an alternative stem cell source for the patients with hematological malignancies requiring allogeneic stem cell transplantation. Cord blood transplantation (CBT) can be performed more quickly than other stem cell transplantation, since cord blood units are preserved in the deep freeze and 1–3 HLA mismatched donors are acceptable. Considering these advantage, we examined the feasibility of cord blood transplantation using reduced-intensity regimens (RI-CBT) for adult relapsed patients after allogeneic tranplantation.

Patients/methods: We reviewed medical records of 26 patients who received RI-CBT at Toranomon Hospital between November 2003 and June 2006. Median age of the patients was 36 years (range, 20–66). Underlying diseases were acute leukemia (n=17), myelodysplastic syndrome (n=4) and lymphoma (n=5). The stem cell source of the first transplantation were bone marrow from sibling donor (n=2), bone marrow from unrelated (n=5) donor, peripheral blood stem cell from sibling donor (n=5) and unrelated cord blood (n=14). Conditioning regimens comprised fludarabine 125–180 mg/m2 in several combination with melphalan 80–140 mg/m2, Busulfan 8–16mg/kg and total body irradiation (TBI) (4–8 Gy). Graft-versus-host disease (GVHD) prophylaxis was cyclosporine (n=5) or tacrolimus (n=21). Median number of total nucleated cells and CD34+ cells was 2.56×106 cells/kg (1.91–5.94), and 0.86×105 cells/kg (0.57–1.77) respectively. HLA disparities were 5/6 (n=2), 4/6 (n=22), and 3/6 (n=2).

Results: Median observation period was 58 days (range, 32–380). Overall survival for 1 year was 15% and 16 patients were died of disease progression (n=5) and infection (n=11). The infection in 4 patients was considered to be caused by regimen related toxicity (RRT). No grade IV toxicities (NCI-CTC Ver.3.0) were observed. The duration between two transplantations was longer in surviving patients compared to dead patients (98 days (range, 39–2108) and 262 days (range, 95–901), respectively), although significant difference was not detected. The stage of the disease in the second transplantation, conditioning regimens and HLA disparities did not influence to the outcome.

Discussion: We demonstrated that RI-CBT could be an available and feasible treatment for the relapsed patients after stem cell transplantation. Moreover, the RRT is acceptable even in the patients with an advanced disease.