bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Effects of Zoledronic Acid on Bone Remodeling.
Authors: Pozzi Samantha , Hideshima Teru , Vallet Sonia , Mukherjee Siddhartha , Chhetri Shweta , Cirstea Diana , Vaghela Nileshwari , Thomas Clare , Rosa Dilani , Ikeda Hiroshi , Okawa Yutaka , Kiziltepe Tanyel , Schoonmaker Jesse , Schipani Ernestina , Bouxsein Mary , Anderson Kenneth C. , Raje Noopur
Published: October 2019

Zoledronic acid (ZA) is an amino-bisphosphonate with very potent antiresorptive activity widely used in the treatment of multiple myeloma (MM) bone disease. Recently, the increasing incidence of osteonecrosis of the jaw and its possible association with prolonged use of amino-bisphosphonates such as ZA has been reported. Therefore, we here studied the effects of ZA on bone remodeling in vitro and in vivo. Although ZA has been shown to inhibit osteoclastogenesis and increase bone mineralization, data on its effects on osteoblastic activity and on bone formation are conflicting. To study the effects of ZA on osteoblasts (OB), 5 week old C57BL/6 mice were treated with low and high doses of ZA intraperitoneally (IP) weekly. The dose ranged from 0.05-1mg/kg, with the highest dose recapitulating a lifetime dose of ZA over a 5 year period in an adult MM patient. Blood was collected at baseline and weekly thereafter. IP calcein injections were administered to study bone formation rates. Consistent with its known effects on bone mass and density, in vivo DXA scans in ZA-treated animals demonstrated an increase in whole body bone mineral density (BMD). ZA treatment was associated with a dose-related increase in trabecular bone at the distal femur, evaluated by microCT and confirmed by static histomorphometry. ELISA assays showed a decrease in TRACP5B (bone resorption), as expected due to the anti-osteoclastic activity of ZA. In addition to osteoclast (OC) inhibition, mice treated with ZA also showed alterations in OB activity. Specifically, serum osteocalcin (a marker for bone formation) levels were lower in ZA treated mice, and dynamic histomorphometry confirmed decreased bone formation rates. In order to study the mechanism of our in vivo observations, we tested the effects of ZA on OB, OC, and bone marrow stromal cells (BMSCs) treated in vitro with ZA (0.01μM to 100μM) for 7, 14, and 21 days. Decreased viability during differentiation of both OB and OC was observed, without any significant effects on BMSCs. This was associated with decreased alkaline phosphatase activity, suggesting functional impairment of OB activity. Our data therefore suggests that ZA impairs OB number and activity, in addition to effects on OC and may impair normal physiologic bone remodeling. Ongoing studies will determine the molecular mechanisms whereby ZA mediates these sequelae and inform future studies of ZA use in patients with MM bone disease.
Binding of TACI and APRIL to Syndecan-1 Confer on Them a Major Role in Multiple Myeloma.
Authors: Moreaux Jerome , Sprynski Anne-Catherine , Mahtouk Karene , Moine Philippe , Robert Nicolas , Rossi Jean-François , Klein Bernard
Published: October 2019

B-cell activating factor (BAFF) and A Proliferation-Inducing Ligand (APRIL) are produced by the bone marrow microenvironment of patients with multiple myeloma (MM) and are growth factors of multiple myeloma cells (MMC). BAFF and APRIL share two receptors - TACI and BCMA - and BAFF binds to a third receptor, BAFF-R. We previously reported that TACI expression is a good indicator of a BAFF-binding receptor in human myeloma cell lines (HMCL), unlike BCMA that is expressed on all HMCL. BAFF-R is lacking. More recently, Ingold et al (J Exp Med; 2005) and Hendriks at al (Cell Death Differ; 2005) identified proteoglycans as the APRIL-specific binding partners. Bischof et al (Blood; 2006) demonstrated that TACI binds also heparan sulfate (HS) chains, in particular to syndecan-1, syndecan-2 and syndecan-4. Syndecan-1 is expressed by plasma cells and epithelial cells and is involved in several cellular processes relying on interactions with extra-cellular matrix proteins, growth factors, chemokines and adhesion molecules. We found a very large binding of APRIL (mean fluorescence activity ≥ 500), unlike BAFF, at the surface of all syndecan-1+ HMCL. These syndecan-1+ HMCL also highly bound TACI-Fc molecules, unlike BCMA-Fc or BAFF-R-Fc molecules. This large binding of APRIL or TACI-Fc molecules was abrogated by heparitinase pretreatment of MMC, removing the HS chains or by preincubation of APRIL and TACI-Fc with heparin. These data were extended to patients primary myeloma cells. In agreement with this large binding to MMC, APRIL was 15 to 40 fold more efficient than BAFF to stimulate the growth of HMCL. The APRIL growth factor activity could be inhibited using heparin, unlike that of BAFF. Our data establish that syndecan-1, by concentrating large levels of APRIL and TACI at MMC surface, can promote APRIL/TACI signaling that induces survival and proliferation of MMC. Heparan sulfate chain inhibitors could be helpful to synergize with BAFF/APRIL inhibitors in MM disease.
The Expression of CD56 Is Frequently Accompanied with the Expression of Neuronal Cell Markers and Its Down-Regulation Is Induced by IL-6 in Human Myeloma Cells.
Authors: Iqbal Mohd S. , Otsuyama Ken-ichiro , Shamsasenjan Karim , Abroun Saeid , Amin Jakia , Islam Abul , Kawano Michio M.
Published: October 2019

Human myeloma cells have the marked phenotypic heterogeneity of surface marker expressions, possibly because of loss of PAX-5 expression. Especially, ectopic expression of CD56, one of non-B cell lineage markers, is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. However, only 2 (NOP2 and AMO1) out of 10 myeloma cell lines were CD56(+). In primary myeloma cells as well as CD56(−) myeloma cell lines, the treatment with forskolin could induce the expression of CD56 in the in vitro culture. In most CD56(+) primary myeloma cells as well as myeloma cell lines, the expressions of neuronal cell markers such as neuron specific enolase (NSE), nestin, β-tubulin III or chromogranin A were found coincidentally. By gene expression profiling, CD56(+) myeloma cell lines showed the marked expressions of transcription factors involved in neuronal cell lineage. On the other hand, addition of IL-6 down-regulated the expression of CD56 in CD56(+) myeloma cell lines in the in vitro culture. In 13 out of 60 patients with overt myeloma, these myeloma cells showed CD56(−) and their values of plasma CRP were significantly increased and MPC-1(−)CD45(+) immature myeloma cells were also increased compared to those in CD56(+) myeloma cases. Therefore, these results indicate that the expression of CD56 is possibly due to phenotypic changes into neuronal cell lineage, and IL-6 can block these phenotypic changes, keeping PAX-5(−) myeloma cells being uncommitted cells to any lineage.
The JAK2 Kinase Inhibitor LS104 Induces Growth-Arrest and Apoptosis in JAK2V617F Positive Cells.
Authors: Lipka Daniel B. , Hoffmann Linda S. , Heidel Florian , Markova Boyka , Kasper Stefan , Breitenbuecher Frank , Kindler Thomas , Levine Ross , Elliot Joseph , Huber Christoph , Fischer Thomas
Published: October 2019

The JAK2V617F-mutation (V617F) is a novel, highly prevalent molecular marker in Ph-negative myeloproliferative disease (MPD). In vitro, the V617F mutation confers cytokine independent growth of Ba/F3 cells expressing erythropoietin receptor (EpoR) and constitutive activation of the JAK2 kinase and of the JAK-STAT pathway. In a murine bone-marrow transplant model the V617F-mutation alone is sufficient to induce a polycythemia vera-like phenotype. Therefore, mutant JAK2 kinase is a promising target for kinase inhibitor development. In this report, we characterize the small molecule LS104 (previously CR4; Grunberger et al., Blood 2003) as a novel non-ATP-competitive JAK2V617F kinase inhibitor. Ba/F3 cells stably transfected with EpoR and either the V617F-mutant (Ba/F3-EpoR-VF) or wildtype JAK2 (Ba/F3-EpoR-WT) were treated with LS104. Apoptosis assays as well as immunoblotting of JAK2 and of downstream signalling pathways were performed. The effects of LS104 on kinase activity were determined in an in vitro JAK2 kinase assay. A combination of LS104 with JAK-inhibitor I, which acts via the ATP-binding site, was tested in apoptosis assays. Finally, growth of endogenous erythroid colonies (EECs) obtained from patients with V617F-positive MPD was tested upon addition of LS104. LS104 selectively and dose dependently induced apoptosis in Ba/F3-EpoR-VF cells as compared to Ba/F3-EpoR-WT control cells. By immunoblotting we found inhibition of JAK2 autophosphorylation and of downstream targets as STAT5, AKT and ERK upon treatment with LS104. Activation of these targets by JAK2 was confirmed in experiments using JAK2 siRNA. The IC50 for JAK2 in an in vitro kinase assay using LS104 was <5μM, and this effect was not reversible using increased ATP-doses. Combination treatment of Ba/F3-EpoR-VF cells using LS104 plus JAK-inhibitor I lead to significantly increased apoptosis as compared to each substance alone. Furthermore, we observed 89% inhibition of in vitro formation of EECs at 10μM LS104 whereas growth of CFU-GM obtained from normal controls was virtually unaffected. Taken together, our data show that LS104 specifically inhibits JAK2 kinase activity and JAK2 downstream signals and thereby induces apoptosis in V617F-positive cells. Our data suggest, that LS104 either irreversibly binds to the ATP-binding site or acts as a substrate kinase inhibitor and may be combined with ATP-competitive JAK2 inhibitors to enhance treatment efficacy. Growth of EECs from patients with MPD is shown to be significantly suppressed by LS104. Based on these data, a phase I/II clinical trial of LS104 for patients with JAK2V617F-positive MPD has been initiated recently.
Choosing the Right Patients for Clinical Trials in Essential Thrombocythemia or Polycythemia Vera: Leukemic or Fibrotic Transformation Risk Assessment among 1061 Patients from a Single Institution.
Authors: Tefferi Ayalew , Gangat Naseema , Wolanskyj Alexandra P. , Schwager Susan M. , Pardanani Animesh , Mesa Ruben A. , McClure Rebecca F. , Li Chin-Yang , Hanson Curtis A.
Published: October 2019

Background: In both polycythemia vera (PV) and essential thrombocythemia (ET), aspirin and/or hydroxyurea therapy provide adequate protection from thrombosis but not from disease transformation into acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) or myelofibrosis (MF). In designing clinical trials with new drugs, including the use of small molecule JAK2 inhibitors, in either ET or PV, it is important to identify patients who are at a higher risk of early transformation into AML/MDS/MF as well as those in whom the long-term risk of these complications is too low to justify exposure to experimental drugs with unknown short-term and long-term toxicity profile.

Methods: Information on an institutional database of 1061 patients with either PV (n=458) or ET (n=603) was updated in July of 2007. The World Health Organization criteria were used for diagnosis of PV, ET, AML, MDS, and MF. Two different approaches were used to assess both early and overall risk of AML/MDS/MF development in both ET and PV. In the first approach, three distinct groups were delineated and their presenting features compared; group A was comprised of patients who have remained AML/MDS/MF free after a minimum follow-up of 20 years whereas groups B and C included patients who developed either AML/MDS or MF, respectively, in the first decade of their disease. In the second approach, prognostic factors for AML/MDS/MF-free survival were examined among the entire cohort of 1061 patients.

Results : In ET, the number of patients that fulfilled the above-mentioned criteria for inclusion in groups A, B, and C were 40, 12, and 8; the three groups displayed significant differences in the prevalence, at diagnosis, of lower than normal hemoglobin level (8% vs. 58% vs. 38%; p=0.0004), male sex (18% vs. 58% vs. 50%; p=0.01), and arterial thrombosis at diagnosis (p=0.03), respectively. However, only the former two remained significant during multivariable analysis that included age as a covariate. In PV, the number of patients in groups A, B, and C were 23, 18, and 12; the only difference between the three groups was the association between group B and leukocytosis (p=0.02). Cox regression analysis of the entire 1061 ET/PV patients cohort, including 65 AML/MDS/MF events in ET and 36 AML/MDS events in PV, confirmed the inferior AML/MDS/MF-free survival in ET associated with lower than normal hemoglobin and AML/MDS-free survival in PV associated with leukocytosis (Figure).

Conclusion : The current study identifies PV patients with leukocytosis as being the most appropriate and ET patients without anemia as the least appropriate for consideration of participation in experimental drug treatment trials.

Figure Figure Figure Figure
Clinical Molecular Diagnosis of T-Cell Receptor Gene Rearrangement in Thai Patients.
Authors: Kummalue Tanawan , Saesue Teerawat , Pongpruttipan Tawatchai , Sukpanichnant Sanya , Opartkiattikul Nisarat
Published: October 2019

Thirteen Thai patients, seven with pathologic diagnosis of T-cell lymphoma, four with clinical and/or pathological suspicion of T-cell lymphoma, one with lymphadenitis and one with chronic hypertrophic tonsillitis, were investigated for T-cell receptor gene rearrangement (TCR-GR) by polymerase chain reaction (PCR). Clonal TCR-GR was demonstrated in each T-cell lymphoma whereas polyclonal TCR-GR was found in the rest of the patients, including the four cases that were suspected of T-cell lymphoma.

Aim: To identify T-cell receptor gene rearrangement in malignant lymphoma in Thai patients.

Material and method: Thirteen specimens of formalin-fixed, paraffin-embedded tissue were collected from Department of Pathology, Faculty of Medicine Siriraj Hospital, Mahidol University. After conventional morphological evaluation and immunohistochemical studies, DNA in each specimen was extracted from 10μm thick paraffin sections using QIAamp DNA mini kit (QIAGEN) for TCR-GR analysis by multiplex PCR. Two pairs of primers were selected for Vγ11 (VγI), Vγ101(VγIII/IV), Jγ12(Jγ1/2) and Jp12(JPγ1/2). DNA of the four cases suspected of malignant lymphoma were repeated by using IdentiClone™ TCRG Gene Clonality Assay kit (InVivoScribe Technologies).

Results: According to the WHO classification (2001), the seven cases of T-cell lymphoma were angioimmunoblastic type (3 cases) and unspecified type (4 cases). All cases showed monoclonal TCR-GR. Only one of the four cases suspected of T-cell lymphoma was doubtful for nasal NK/T-cell lymphoma while the others were suspected clinically. All of these four cases and the two reactive conditions revealed polyclonal TCR-GR. After using IdentiClone™ kit twice, the four cases suspected of T-cell lymphoma still showed polyclonal TCR-GR.

Conclusion: The molecular analysis of TCR-GR performed in thirteen cases demonstrated the monoclonal TCR-GR in all seven cases of T-cell lymphoma and polyclonal TCR-GR in the four cases suspected of T-cell lymphoma and the two cases of reactive condition. Therefore, this technique is very useful to confirm the diagnosis of T-cell lymphoma and to provide information for further management in patients suspected of malignant lymphoma.
Early Relapses in Primary CNS Lymphoma (PCNSL) without Intraventricular Treatment.
Authors: Schmidt-Wolf Ingo G.H. , Pels Hendrick , Jurgens Annika , Glasmacher Axel , Schulz Holger , Engert Andreas , Linnebank Michael , Schackert Gabriele , Reichmann Heinz , Kroschinsky Frank , Vogt-Schaden Marlies , Egerer Gertlinde , Schaller Carlo , Lamprecht Monika , Bogdahn Ulrich , Deckert Martina , Fimmers Rolf , Bangard Christoph , Schlegel Uwe
Published: October 2019

Background: A systemic and intraventricular polychemotherapy regimen (“Bonn protocol”) with deferred radiotherapy had resulted in durable responses in 75% of patients < 60 years with primary CNS lymphoma (PCNSL), but had been complicated by a high rate of Ommaya reservoir infections.

Purpose: Here, efficacy and toxicity of this regimen but without intraventricular treatment was evaluated in PCNSL.

Patients and Methods : From 08/03 to 11/05, 18 patients with PCNSL < 60 years (median age 53 years) were treated within a phase II trial with a high-dose methotrexate (MTX; cycles 1,2,4 and 5) and cytarabine (Ara-C; cycles 3 and 6) based systemic therapy including dexamethasone, vinca-alkaloids, ifosfamide and cyclophosphamide.

Results: Study accrual was prematurely stopped in 11/05 due to a high rate of early relapses. Seventeen/18 patients were assessable for response: Nine (53%) achieved complete response (CR), two (12%) complete response/unconfirmed (CRu), two (12%) partial response (PR), four (24%) showed progressive disease (PD); in one treatment was stopped due to toxicity. Median follow-up is 23 months; Kaplan-Meier estimates for median response duration were ten months only in responding patients and for median time to treatment failure (TTF) eight months in the whole group; median overall survival (OS) has not yet been reached. Systemic toxicity was mainly hematologic.

Conclusions: In patients < 60 years with PCNSL polychemotherapy without intraventricular treatment results in a high response rate, but is associated with early relapses in the majority of cases. This is in contrast to the results achieved with the same protocol but with inclusion of intraventricular treatment.
Addition of Rituximab to CHOP Chemotherapy Does Not Improve Clinical Outcome in Patients with Localized Gastric Diffuse Large B-Cell Lymphoma: A Retrospective Study by Ganken Adult Lymphoma Study Group (Single Institute Analysis).
Authors: Yokoyama Masahiro , Ennishi Daisuke , Asai Hiroaki , Ueda Kyoko , Kodaira Makoto , Yamada Shuhei , Myojo Tomohiro , Mishima Yuko , Sakajiri Sakura , Takeuchi Kengo , Saotome Takashi , Terui Yasuhito , Takahashi Shunji , Ito Yoshinori , Hatake Kiyohiko
Published: October 2019

Background: In recent years, it has been reported that addition of rituximab to CHOP chemotherapy significantly improves event-free survival (EFS) and overall survival (OS) compared with standard CHOP alone in patients with diffuse large B-cell lymphoma (DLBCL). On the other hand, gastric DLBCL is a good clinical outcome without surgical excision in the group of primary extranodal DLBCL. Localized gastric DLBCL shows very good EFS and OS treated with standard CHOP plus radiation therapy without rituximab.

Methods: We analyzed retrospectively the prognosis between R-CHOP and CHOP alone in patients with localized gastric DLBCL. All 24 patients diagnosed between July 1999 and July 2006 in our hospital, were included in this study. The pathology was reviewed by a hematopathologist and confirmed to be de novo DLBCL according to the WHO classification. The staging system was using by Lugano meeting classification. Patients with stage IIE and IV were excluded. All patients were initially treated with six cycles of CHOP or R-CHOP regimen consisting of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone. If we evaluated partial response after six cycles of chemothrapy, the patients have been added radiation therapy.

Results: Initial characteristics of the 24 patients are summarized in Table 1. The median age of patients at time of inclusion was 67.5 yr (range 30–83 yr). The median follow up of surviving patients was 30 months. CR rates between CHOP and R-CHOP were 91.7% vs. 83.3%, p=1.000, and overall response rates were 100% vs. 83.3%, p=.478, respectively. 2 years progression-free survival and overall survival were no statistical significance difference between two groups (Log rank test: P=.138, P=.739).

Conclusion: These results suggested that addition of rituximab to CHOP chemotherapy does not improve clinical outcome of localized gastric DLBCL. The patients with localized gastric DLBCL did not get the clinical benefits of rituximab. As you know, rituximab needs high costs, so we recommend standard CHOP regimen plus radiation therapy in patients with localized gastric DLBCL, without rituximab combination regimen.

Characteristics of the 24 patients with localized gastric DLBCL Characteristic CHOP (n=12) R-CHOP (n=12) P Age 1.000 =<60 3 4 >60 9 8 Male 7 6 1.000 Stage (Lugano) .890 I 4 3 II 1 7 9 Unknown 1 0 IPI score .867 0,1 7 7 2 4 5 Unknown 1 0
Phase I Study of KW-0761, a Humanized Anti-CCR4 Antibody, in Patients (Pts) with Relapsed or Refractory Adult T-Cell Leukemia-Lymphoma (ATLL) and Peripheral T-Cell Lymphoma (PTCL): Preliminary Results.
Authors: Uike Naokuni , Tsukasaki Kunihiro , Utsunomiya Atae , Tobinai Kensei , Morishima Yasuo , Uozumi Kimiharu , Tomonaga Masao , Matsushima Koji , Shitara Knya , Akinaga Shiro , Ueda Ryuzo
Published: October 2019

Introduction: KW-0761 is a defucosylated humanized IgG1 monoclonal antibody against CC chemokine receptor 4 (CCR4). Previous studies revealed that CCR4 was over-expressed on tumor cells from 88% of pts with ATLL and 38% of pts with PTCL. CCR4 expression was associated with unfavorable prognosis in both diseases. These evidences suggest that CCR4 could be a reasonable molecular target for treatment of CCR4-positive ATLL and PTCL. KW-0761 exerted very potent ADCC but not CDC against cultured ATLL cell lines using normal effecter cells. KW-0761 also exhibited potent ADCC against freshly isolated ATLL cells using the auto-effecter cells.

Methods: KW-0761 is being investigated in a Phase I, single-agent, dose-escalation, multicenter study for relapsed or refractory pts with ATLL or PTCL after undergoing the initial chemotherapy. Eligible pts were required to express CCR4 on their tumor cells by FCM and/or IHC. This phase I trial was designed to evaluate toxicity, pharmacokinetics (PK), immunogenicity and anti-tumor activity. Toxicity was evaluated by NCI CTCAE (v3.0). Pts were planned to receive four weekly intravenous injections of KW-0761 at the doses of 0.01, 0.1, 0.5 and 1.0 mg/kg.

Results: As of August 10, 2007, 6 pts (1 PTCL and 5 ATLL) have been treated with KW-0761 at the dose of 0.01 (N=3) and 0.1 mg/kg (N=3). Three pts were evaluable for toxicity, PK, immunogenicity and 4 pts were evaluable for anti-tumor activity. KW-0761 was well tolerated with no dose-limiting toxicities. Reversible grade (G) 3 toxicities were lymphocytopenia and herpes zoster. G1-2 adverse events were; rash, constipation, nausea, EF decrease, neutropenia, thrombocytopenia, eosinophilia, lymphocytopenia, ALP increase and QT prolongation. In one leukemic ATLL pt with swollen lymph nodes (LNs) treated at 0.01 mg/kg, peripheral ATLL cells disappeared and the LNs were decreased in size, attaining PR. In another leukemic ATLL pt with skin involvement treated at 0.1 mg/kg, peripheral ATLL cells disappeared and the skin achieved PR by Physicians Global Assessment of Clinical Conditions (PGA). One ATLL pt with skin and bone involvement at 0.01 mg/kg showed SD. Preliminary PK analysis at 0.01 mg/kg showed that Cmax and T1/2 after the 4th dosing was 323.7 56.7 ng/ml and 244 117 hrs, respectively. KW-0761 did not exhibit immunogenicity at 0.01 mg/kg.

Conclusions: Preliminary phase I data warrant further investigations of KW-0761, a first-in-class humanized anti-CCR4 antibody, against CCR4-positive peripheral T-cell malignancies including ATLL. Patient accrual is ongoing and the updated results will be presented at the meeting.
Detecting Apoptotic Blocks and Sensitvity to ABT-737 and Conventional Chemotherapy Via BH3 Profiling.
Authors: Letai Anthony , Shipp Margaret , Cin Paola dal , Takeyama Kunihiko , Deng Jing
Published: October 2019

Cancer cells require blocks in apoptosis to survive despite many abnormalities that should trigger apoptosis. Here we demonstrate that BH3 profiling is an effective tool for the systematic investigation of the types of blocks in apoptosis for which cancer cells select. BH3 profiling can segregate lymphoma cell lines into three different classes based on the type of apoptotic block each exhibits. In one class, cells are kept alive via expression of anti-apoptotic proteins of the BCL-2 family. When BCL-2 itself is detected by BH3 profiling as the protein keeping the cell alive, the cell is sensitive to ABT-737, a BCL-2 antagonist compound. Sensitivity to ABT-737 correlates highly with abundance of pro-apoptotic BIM complexed to BCL-2. Knockdown of BIM reduces sensitivity to ABT-737. We describe such cancer cells that maintain survival based on tonic sequestration of large amounts of pro-death molecules by anti-apoptotic proteins like BCL-2 as “primed for death”. “Primed” lymphoma cell lines are more sensitive than “unprimed” cancer cells to conventional agents killing via apoptosis like vincristine, adriamycin, and etoposide. It is important to note that, in general, non-malignant cells are unlikely to be “primed”. Hence “priming” may be an important determinant of the therapeutic window between normal and malignant cells. These results suggest that the degree to which a cell is “primed” can influence sensitivity to chemotherapy agents targeting distinct cellular targets. Therefore, BH3 profiling is a tool potentially useful not only for predicting sensitivity to agents targeting antiapoptotic BCL-2 proteins, but also to conventional cytotoxic agents that use the mitochondrial apoptotic pathway to kill.
The Feature of TCR Zeta Gene in CD4+ and CD8+ T Cells in Patients with CML.
Authors: Chen Si , Li Yangqiu , Chen Shaohua , Yang Lijian , Wu Xiuli , Cen Dongzhi
Published: October 2019

The T-cell receptor (TCR) zeta chain is a master sensor and regulator of lymphocyte responses which plays a critical role in TCR-mediated signal transduction. The abnormal expression of TCR zeta gene was found in some malignancies and immune disease. Cellular immune deficiency is the common feature in patients with chronic myeloid leukemia (CML). Our previous studies had showed that 60% of CML patients displayed an abnormally low expression of TCR zeta chain in peripheral blood mononuclear cells (PBMCs). To further estimate the changes of zeta chain expression of T-lymphocyte subsets in CML, TCR zeta chain gene expression level in purified CD4+ and CD8+ T cells sorted by MACS were detected by real-Time PCR with SYBR Green I technique, CD4+ and CD8+ T cells from both 10 cases with CML and 10 normal individuals were selected to analyze in the study. β2 -microglobulin was used as an endogenous reference. Relative changes in TCR zeta gene expression level were used by the 2−ΔCt×100% method. The relative mRNA expression level of TCR zeta gene in CD4+ and CD8+ T cells from CML was 4.06±4.82% and 3.82±4.25% respectively, whereas 7.75±2.52% and 5.19±1.25% were found in normal individuals. There was a wide range in the TCR zeta gene expression level of CD4+ T cells from CML patients (0.23–15.93%,) with a median value of 1.77%, whereas the a range of (4.74–12.81%) with a median of 7.93% was showed in normal controls (P=0.046). The expression level of TCR zeta gene in CD8+ T cells from controls ranged from 3.47% to 6.75% (median 5.52%) and in CML patients from 0.86% to 15.55% with a median of 2.51% (P=0.340). The results indicated that, compared with normal controls, zeta chain gene expression was obviously down-regulated in CD4+ T cells from patients with CML. However, there was not significant different in expression level of TCR zeta gene of CD8+ T cells between CML patients and normal individuals. Moreover, the expression level of TCR zeta gene is nonsignificant age-associated or gender-link in CD4+ T cells and CD8+ T cells from patients with CML. In conclusions, the results provide at first the difference of TCR-zeta gene expression pattern in CD4+ and CD8+ T cells from patients with CML, this may be reflective of signaling difference between the different T cell subtypes in immunodeficiency state of patients.
Bcl-6 May Be a Survival Factor in Pre-B Cell Ph+ Blast Crisis Cell Lines.
Authors: Batlle Ana , Lam Eric W. , Wagner Simon D.
Published: October 2019

The constitutively active tyrosine kinase Bcr-Abl fusion protein is essential for the development of chronic myeloid leukaemia. Inhibitors of its tyrosine kinase activity e.g. Imatinib, are highly effective in treating chronic phase disease but are only transiently useful in blast crisis, especially lymphoid blast crisis. Bcl-6 is a transcriptional repressor that is required for the formation and maintenance of germinal centre B-cells. Following reports that Imatinib increases expression of Bcl-6 in Ph+ cell lines representative of lymphoid blast crisis we have investigated the regulation of this molecule and its functional importance. We utilised BV173 and Z119. Basal Bcl-6 protein expression was detectable in Z119, but not BV173. As anticipated Imatinib increased Bcl-6 expression in both cell lines. STAT5 is an important target of Bcr-Abl and has been implicated as both a positive and negative regulator of Bcl-6 transcription. Transient transfection with a mammalian expression plasmid bearing a dominant negative STAT5 reduced Bcl-6 expression in Z119 implying that activated STAT5 promotes Bcl-6 expression in this cell line, but also that STAT5 cannot be the main target of Bcr-Abl because Imatinib and dominant negative STAT5 have opposing effects. Next we considered the possibility that phosphorylation status of the transcription factor FoxO3a, which has been shown to bind to the Bcl-6 promoter, correlated with Bcl-6 expression. We found that decreased phosphorylation of Foxo3a, activating this transcription factor, was highly associated with increased Bcl-6 expression suggesting that it is an significant Bcr-Abl target. Next we wished to find out the functional implications of increased Bcl-6 expression. We found that a low concentration of Imatinib (0.25μM) was sufficient to induce Bcl-6 in BV173 and Z119, and caused death of these cell lines over several days. Next we combined Imatinib with a previously reported peptide antagonist of the Bcl-6/SMRT co-repressor interaction. Neither this peptide nor a mutated negative control peptide had an effect on cell numbers or apoptosis over 48 hours of culture when used alone. However, when combined with Imatinib the wild-type peptide, but not the control, reduced cell growth in Z119. We conclude that Bcl-6 may be a survival factor in Ph+ lymphoid blast crisis cell lines following treatment with Imatinib and that specific treatments to abrogate Bcl-6 function may find a place in the treatment of lymphoid blast crisis of CML.
Cardiotoxicity of Imatinib Mesylate - A Prospective Cross-Sectional Study.
Authors: Bittencourt Henrique , Marcolino Milena S. , Clementino Nelma , Xavier Vitor F. , Barbosa Marcia M. , Nunes Maria do Carmo P. , Ribeiro Antonio L.
Published: October 2019

Imatinib Mesylate (IM), a tyrosine kinase inhibitor, has become the first line of therapy for patients with chronic myeloid leukemia (CML) and the standard treatment of advanced gastrointestinal stromal tumors (GIST). A recent publication (Kerkela et al. Nature2006;12:908) suggest that IM might have myocardial toxicity via c-Abl inhibition. Retrospective studies, however, do not show an increase of cardiac hearth failure (CHF) in patients on IM therapy in clinical trials for CML or GIST (Atallah et al. Blood2007;110:1233 and Verweij et al. Eur J Cancer2007; 43:974). In order to prospectively evaluate IM cardiotoxicity we included 101 patients with CML on treatment with IM. As control group, we also included 57 patients with myeloproliferative disorders not treated with IM (essential thrombocytemia= 27, CML=12, policitemia vera=11, other diagnosis=7). After informed consent, all patients had a complete clinical evaluation (interview and physical examination), blood samples for Brain Natriuretic Peptide (BNP) were taken and an echocardiographic study was performed. Groups were similar regarding age (median 48 vs. 54 years), gender distribution and cardiac risk factors, except for hypertension (lower prevalence in IM group: 26% vs. 45.6% in controls, p<0.05). Median time of IM treatment was 805 (range 50–2122) days, mean and median actual IM dose were 463mg and 400 mg, respectively. Cardiac symptoms and signs were equally distributed between groups, except for peripheral edema, more frequent in IM group (25% vs 5%, p<0.05). There was no statistical difference regarding BNP levels (24.4± 7.7pg/mL vs. 26.5± 6.7pg/mL), end diastolic volume of left ventricle (49.4± 10.3mL vs. 48.0 ± 10.8mL) and ejection fraction (EF, 69± 16% vs. 68±13%) for IM and control group, respectively. Four patients presented BNP level above the upper normal limit (UNL = 100pg/mL) in IM group and none in control group. There was a trend for an association between BNP above UNL and a higher IM actual dose (P=0.08). Two patients in IM group and none in control group presented an ejection fraction below normal (<55%). This study shows that a systematic deterioration of cardiac function mediated by IM therapy was not observed. However, since some patients presented with alteration of BNP and/or EF, there is still a possibility for isolate cardiotoxicity associated with IM. BNP, a simple test to perform, might be use to identify patients at risk for subsequent CHF while on IM therapy.
Low Dose and Standard Dose of Imatinib Therapy for Patients with Chronic Myeloid Leukemia in Akita Prefecture, Japan.
Authors: Takahashi Naoto , Kameoka Yoshihiro , Saitoh Hirofumi , Fujishima Naohito , Hirokawa Makoto , Sawada Kenichi
Published: October 2019

The IRIS study for CML patients demonstrated the excellent clinical and cytogenetic/molecular effects of imatinib. Patients participating in the IRIS trial were selected according to strict eligibility criteria. The clinical features of patients are usually much more heterogeneous in practical situations than in clinical trials. Sometimes, patients cannot be treated with the standard dose of imatinib because of severe toxicity, especially older patients or patients who had already been treated with the other drugs. In this study, we analyzed whether patients could still be effectively treated using lower doses. Our study analyzed 86 CML patients from 17 hospitals in Akita prefecture. 80 patients were in CP, one patient was in AP, 4 patients were in BC, and one patient had cytogenetic relapse after allo-SCT. All patients were treated with imatinib between December 2001 and July 2007. Initially a dose of 400mg/day was given to almost all patients. Later the dose was decreased in a subset of patients experiencing imatinib-induced side effects. We classified patients into two groups according to the imatinib dosage and analyzed their clinical characteristics [Table 1] and the accumulation of CCR/MMR [Figure 1, 2]. In Group 1, we analyzed 55 patients received 300mg or more of imantiib per day. In Group 2, we analyzed 31 patients received less than 300mg of imatinib per day. Patients in Group 2 were older and had more histories of pretreatment and showed a higher frequency of adverse effects of imatinib than in Group 1. There were no significant differences of CCR/MMR rate between Group 1 and Group 2. This study reproduces the imatinib efficacy results described in the IRIS study, not only for patients treated with standard dose imatinib and for patients who could not take 400mg/day because of imatinib toxicity or other complications. We did not observe an increase in frequency of BCR-ABL point mutations in patients receiving a lower dose of imatinib, suggesting that the low dose imatinib treatment analyzed in our study does not enhance imatinib resistance by increasing BCR-ABL point mutations. In conclusion, we provide data supporting the use of lower doses of imatinib for CML patients that cannot be given sufficient dosage of imatinib for reasons such as severe hematological or non-hematological side effects or other complication.

Clinical Characteristics of Patients in Group I and II Group 1(n=55) Group 2(n=31) P Average doseage 380 mg/day 185 mg/day Average age (% of over 70 yrs) 57.7 yrs (22 %) 68.0 yrs (58 %) .001 (.007) History of treatment with IFN/HU before imatinib therapy 16% 55%. 002 Adverse effect (Grade3/4) 16% 42% .009 PFS to AP/BC at 60 Mo 97.8% 89.6% .28 CCR /MMR rate 78% / 51% 61% / 39% .09 / .27 Point mutation of bcr-abl in patients without MMR 4/17 2/13 .99 Figure Figure Figure Figure
Importance of JAK2 V617F Allele Burden in the Diagnosis of Myeloproliferative Diseases and Its Association to Age.
Authors: Rapado Inmaculada , Albizua Enriqueta , Ayala Rosa , Hernandez Jose Angel , Garcia-Alonso Luis , Grande Silvia , Gallardo Miguel , Gilsanz Florinda , Martinez-Lopez Joaquin
Published: October 2019

The aim of this study is to compare the diagnostic value of several methods, with different sensitivities, to detect JAK2 V617F mutation. Three new methods based on Real Time PCR to detect JAK2 V617F mutation were employed. The first two, based on hybridization probes and Peptide Nucleic Acid Probe (PNA) specific for wild allele; and the third employing Allele Specific Oligonucleotide (ASO) primers for JAK2 V617F mutant allele and MGB TaqMan probe. Healthy subjects (n 149) and patients with Essential Thrombocythemia (ET) (n 63), Polycythemia Vera (PV) (n 31), Secondary Thrombocythosis (ST) (n 38), Secondary Erytrocythosis (SE) (n 35) and other haematological malignancies (n 72) were included in the study. Results. Validity test study for JAK2 617 using HP PCR showed: A-. For PV patients: Sensitivity (Se) was 87.1%, Specificity (Sp) 100%, Positive Predictive value (PPV) 100%, and Negative Predictive Value (NPV) 97.9%. B-. For ET patients: Se was 61.3%, Sp 100%, PPV 100%, and NPV 93.9%. With JAK2 617 PNA PCR: A.- For PV patients: Se was 93.6%, Sp 97.8%, PPV 87.9%, and NPV 98.9%. B. - For ET patients: Se was 71%, Sp 95.7%, PPV 73.3%, and NPV 95.2%. With JAK2 V671F ASO quantitative PCR (JAK2 617 ASO qPCR): A.- For PV patients: Se was 93.5%, Sp 98.5%, PPV 90.6%, and NPV 98.9%. B.-For ET patients: Se was 80.6%, Sp 95.9%, PPV 75.8%, and NPV 96.7%. Cutoff point of 1% was established by ROC curves as mutation burden to distinguish PV or ET versus secondary causes. Three healthy subjects were positive (2%, 3/149) by JAK2 617 PNA PCR method. With JAK2 617 ASO qPCR, 16 healthy subjects would be positive (10%) if cutoff burden established in 0.1%. Significant differences were obtained when comparing Myeloproliferative Disease (MPD) patients over 40 years (n 64) to patients under 40 (n 15). JAK2 V617F median allele burden was 18.41% and 7.26% respectivily (p=0.016).

Conclusions: JAK2 617 HP and ASO qPCR are the best tests in differential diagnosis of Polyglobulias and Thrombocytosis. When JAK2 V617F allele burden is low, the best test for MPD diagnosis is JAK2 617 ASO qPCR. The number of JAK2 V617F mutated cells is higher in patients over 40, which leads to think that age may influence in tumour burden.
Bleeding Risk in Patients with Essential Thrombocythemia and a Platelet Count of More Than 1.5 Million/μl: A Challenge of the Dogma of Mandatory Initiation of Cytoreductive Therapy.
Authors: Petrides Petro E. , Siegel Fabian
Published: October 2019

Background: The most important limiting factors of patient survival in essential thrombocythemia (ET) are thromboembolic and hemorrhagic complications [1]. It has been postulated that the paradox of hemorrhagic events in a disease with highly elevated platelet counts is caused by an acquired von Willebrand Factor (vWF) deficiency syndrome which is found in ET patients with very high platelet counts [2]. Therefore, cytoreductive therapy is usually recommended by several international expert groups when platelets exceed 1,5 Million/μl.

Aims: To critically examine the concept of cytoreductive therapy in high-platelet ET based upon the postulated increased bleeding risk.

Methods: In a prospective study we observed 3 patients with a continously elevated platelet counts of more than 1.5 million/μl and secondary loss of large von Willebrand factor multimers, as shown in SDS electrophoresis, that did not receive cytoreductive therapy, but have been treated only with aspirin.

Results: Patient 1 is a female which was 48 years at the time of diagnosis. She is negative for the JAK2V617F mutation and shows a secondary loss of large vWF multimers. We have now observed her for 7 years without any bleeding complications on aspirin therapy. During that period she has had more than 1.5 million platelets/μl for more than 46 months with a zenith of 2.27 million/μl. The second patient is a male who was 60 years old at the time of diagnosis. He is JAK2V617F negative and shows a relative decrease in large vWF multimers. He has been bleeding free since his diagnosis 6 years ago and had platelet levels of more than 1.5 million/μl for a cumulative period of more than 30 months. He has been treated only with aspirin. Patient 3 is a 59 year old male who has been diagnosed with ET 8 months ago. He is heterozygous positive for the JAK2V617F mutation. He also has a relative decrease in large multimers including a complete loss of the largest multimers. His platelet count has been elevated over 1.5 Million/μl (maximum: 2.18 Million) for a cumulative period of 6 months during which he has been treated only with aspirin. After that period he has been switched to anagrelide due to headaches. He has not shown any hemorrhagic complications except an increased bleeding tendency during shaving.

Conclusion: Our observations challenge the recommendations by several groups to automatically initiate platelet reducing therapy above a platelet count of 1.5 million/μl. Acquired von Willebrand factor deficiency caused by ET does apparently not lead to hemorrhagic complications in all patients and therapy with aspirin should not be automatically excluded based on this dogma.
T Cell Pathway Deficiencies in Chronic Lymphocityc Leukemia: Partial Restoration with OKT3/IL-2 Activation.
Authors: Di Ianni Mauro , Moretti Lorenzo , Del Papa Beatrice , De Ioanni Maria , Terenzi Adelmo , Bazzucchi Moira , Ciurnelli Raffaella , Falzetti Franca , Tabilio Antonio
Published: October 2019

As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p<0.05) in CD3+ cells from CLL patients compared with healthy donors. Panther software analysis identified 34/237 upregulated genes and 26/221 downregulated genes that were involved in specific pathways, mainly cell differentiation and proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T cell activation. The 26 dowregulated genes included Zap70, a member of the syk family protein tyrosine kinase, which is involved in T-cell activation. Zap-70 results were validated by mRNA quantification by RT-PCR (−1.77 fold in comparison with healthy controls) and by flow-cytometric analysis (Mean Intensity Fluorescence=33±12 vs 80±23.62 in controls, p<0.05). To test the hypothesis that activation with OKT3 /IL-2 could bypass these T cell deficiencies, activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity (using the 51chromium release assay) against mutated and unmutated (according to IgVH mutational status) autologous B cells, DAUDI, K562 and P815 cell lines. After 10 days’ culture, the T cell count remained unchanged; CD8 cells expanded more than CD4; TCR spectratyping analysis indicated no differences in TCR repertoires. Activation restored the ZAP-70 mRNA (+1.67 fold). The 51chromium release cytotoxicity assay showed an index > 30% in 5/20 patients. The other 15 were partially cytotoxic against P815, K562 and Daudi. Cell line analysis in all 20 confirmed prevalently T cell-mediated cytotoxicity and poor NK/LAK activity. Cytotoxicity did not correlate with B cell mutational status. We tested the cytotoxic activity of autologous activated T cells in NOD/SCID mice co-transplanted with leukaemic B cells. Only activated T cells exerting cytotoxicity vs autologous B-cell CLL prevent CLL in human-mouse chimera, as confirmed by PCR and FACS analysis which visualised only CD3+ cells. In conclusion, in patients with CLL, activating autologous T cells with OKT3 /IL-2 bypasses, at least in part, the T cell immunological deficiencies. These in vitro and in vivo findings might serve to throw light on new mechanisms that could be exploited in immunotherapy designed to exert disease control.
Antigen Activation and Impaired Fas-Induced Death-Inducing Signaling Complex (DISC) Formation in Large Granular Lymphocyte Leukemaia.
Authors: Yang Jun , Epling-Burrbette P.K. , Painter Jeffrey S. , Zou Jian , Bai Fanqi , Wei Sheng , Loughran Thomas P.
Published: October 2019

Clonal T-cell expansion in patients with T-Large-Granular Lymphocyte (LGL) Leukemia occurs by an undefined mechanism that may be related to Fas apoptosis resistance. Here we demonstrate polarized expansion of CD8+ terminal-memory differentiation in such patients, as demonstrated by CD45RA expression and absence of CD62L expression, suggesting repeated stimulation by antigen in vivo. Elimination of antigen-stimulated T-cells normally occurs through Fas-mediated apoptosis. We show that cells from LGL leukemia patients express c-FLIP and display resistance to Fas-mediated apoptosis and abridged recruitment of proteins that comprise the Death Inducing Signaling Complex (DISC) including the Fas-associated death-domain (FADD) protein and caspase-8. Exposure to interleukin-2 (IL-2) for only 24 hours sensitized leukemic LGL to Fas-mediated apoptosis with enhanced formation of the DISC, and increased caspase-8 and caspase-3 activities; a process that required antigen stimulation in normal T-cells. Our results demonstrate that expanded T-cells in patients with LGL leukemia display both functional and phenotypic characteristics of prior antigen activation in vivo and display reduced capacity for Fas-mediated DISC formation.
Early Reduction of Serum Free Light Chain Can Predict Therapeutic Responses in Multiple Myeloma.
Authors: Nakorn Thanyaphong Na , Watanaboonyongcharoen Phandee , Suwannabutra Seksan , Theerasaksilp Sunee , Paritpokee Nara
Published: October 2019

Background M protein as measured by serum protein electrophoresis (SPE) is usually required for the diagnosis and therapeutic monitoring of multiple myeloma. However, SPE may not be able to detect M protein in some cases such as light-chain disease or non-secretory myeloma. Serum free light chain (FLC) assay has been recently developed and shown to be very helpful in such situations. Here, we evaluate the usefulness of this new test in the diagnosis and therapeutic monitoring of myeloma patients treated with bortezomib plus dexamethasone at our institution.

Methods Serial serum FLC assays were performed before and after each cycle of induction therapy. All patients were treated with bortezomib plus dexamethasone every 3 weeks for four cycles. Treatment responses were determined according to the European Group for Blood and Marrow Transplantation criteria.

Results There were total of 25 patients in this analysis. Fifteen were male (60%); the median age was 54 years (range, 35–68 years). Abnormal ratio of serum FLC was detected in 88% of pre-treatment serum samples. After four cycles of induction therapy, nine patients (36%) had achieved complete remission. No baseline clinical and laboratory parameters could correctly predict the therapeutic responses in these patients. When serial FLC assays were taken into account, we found that a more-than-40% reduction in the abnormal serum FLC after the first cycle of treatment was significantly associated with achieving complete response at the end of treatment (Pearson Chi-square, p = 0.041).

Conclusions Serum FLC assay is very useful for the diagnosis of multiple myeloma and allows a more rapid assessment of therapeutic response. Further studies are needed to establish the role of this test in the era of highly effective anti-myeloma therapy.