bluuebunny/crossref_metadata_2025 huggingface dataset:
Megakaryocyte-Active Chemokines: Dysregulation in the SDF-1a/CXCR4 Axis in Patients with Essential Thrombocythemia.
Authors: Salim Juan P. , Marta Rosana F. , Molinas Felisa C.
Published: April 2020

Chemokines belong to a large family of molecules that are implicated in the localization and production of blood cells. Some of them, such as Interleukin 8 (IL-8) and GRO-a, participate in the regulation of megakaryopoiesis, mostly by exerting an inhibitory action. Recently, the involvement of stromal derived factor 1 (SDF-1) in synergizing the stimulatory effect of thrombopoietin on megakaryopoiesis and its participation in megakaryocyte transendothelial migration has been described. The aim of the present study was the evaluation of the plasma levels of IL-8, GRO- a and SDF-1 in patients with essential thrombocythemia (ET), a myeloproliferative disorder characterized by megakaryocytic hyperproliferation with increased circulating platelet count. Besides, the corresponding chemokine receptors were assayed on platelet membrane from ET patients. A cohort of 27 patients diagnosed according to the Polycitemia Vera Study Group were enrolled in the study (mean age, 45; 21 women). Twenty-seven normal subjects matched by sex and age were taken as the control group. The Ethic Committee from IDIM A. Lanari approved the study and all patients and normal controls signed the informed consent. Plasma levels of the chemokines were measured by ELISA technique (R&D Systems) according to the manufacturer. Expression levels of IL-8 receptors (CXCR1 and CXCR2), GRO-a receptors (CXCR2) and SDF-1 receptors (CXCR4) on platelet membrane were evaluated by flow cytometry using specific MoAbs and the corresponding isotype controls (B-D Pharmingen). Flow cytometry results were expressed as relative fluorescence intensity (RFI, the relationship between the mean fluorescence intensity from the specific antibody and the isotype control). Results were expressed as median and range. Statistic analysis was carried out using Mann-Whitney Wilcoxon rank sum test and Wilcoxon signed rank test. Plasma levels of the chemokines measured in 19 ET patients were similar to that found in normal controls, IL-8 2.5, pg/ml (0.8–28.2) and 2.8 pg/ml (1.1–16.5), GRO-a, 30.0 pg/ml (7.4–463.1) and 23.9 pg/ml (9.6–148.0), SDF-1, 1895.0 pg/ml (1246.0–2719.0) and 1915.0 pg/ml (822–2424.0), respectively. The expression levels of CXCR4 receptor was found diminished in platelets from ET patients, RIF 16.94 (1.3–31.3) compared to normal controls, 27.4 (2.4–58.4); p=0.0059, n=10. However, the expression of CXCR1 and CXCR2 in platelets from ET patients was normal. In conclusion, although plasma levels of the chemoquines IL-8, GRO-a and SDF-1 were normal in these patients, the decreased level of CXCR4 on platelet membrane suggests a dysregulation in the SDF-1a/CXCR4 axis in patients with essential thrombocythemia.
A Cautionary Tale.
Authors: Pearlman Eugene S. , Mitchell Farzine , Carloss Harry
Published: April 2020

RV, a 62 year old caucasian male was admitted to LH from the ER for prolonged epistaxis. Ten months prior to admission he had appeared at the ER with the same complaint. At that time the patient’s Hb, Hmct, MCH and MCHC were 12.3 g/dL, 29.2 percent, 35.8 pg and 42 percent respectively. All hematological and chemical assays used instrumentation from Beckman-Coulter Inc.( Brea, CA). The patient’s epistaxis was attributed to anti-platelet therapy with aspirin and clopidogrel for coronary artery disease and he was subsequently lost to follow-up. At his most recent ER appearance his Hb, Hmct, MCH and MCHC were 11.8 g/dL, 22.9 percent, 42.5 pg and 51.6 percent respectively. The patient was admitted to allow evaluation by ENT because of difficulty controlling his epistaxis. Shortly after admission, a CBC revealed nearly identical hematological parameters to those seen in the ER and the pathologist was telephoned. Attempts at warming the patient’s specimen and even acquiring another specimen brought to the lab in a water bath resulted in only trivial changes in the patient’s CBC. If however, patient plasma was replaced by an equal volume of isotonic diluent supplied by the manufacturer, the Hb, Hmct, MCH and MCHC dropped to 6.5 g/dL, 19.4 percent, 23.1 pg and 33.5 percent respectively. Examination of a smear revealed numerous rouleaux. Serum protein and albumin assays revealed concentrations of 9.7 and 2.7 g/dL respectively suggesting an increase in the globulin fraction. Subsequent SPE, IFE and quantitative nephelometry assays performed at our reference lab (ARUP; Salt Lake City, UT) indicated the presence of an IgM-kappa monoclonal protein [6.99 g/dL]. A presumptive diagnosis of Waldenstrom’s macroglobulinemia was confirmed by bone marow biopsy. It has long been known that an MCHC greater than 36 percent should prompt a careful investigation of the data [Am J Clin Pathlol.83:218 (1985)]. In this case the elevated concentration of IgM resulted in increased turbidity and a spuriously elevated Hb [Arch Pathol Lab Med.124:616 (2000)]. Yet the clue went unrecoginized by laboratory personnel as well as ordering physicians leading to a probable delay of nearly one year in the diagnosis of the patient’s underlying hematologiclal disorder.
CD4+ Perforin+ T Cells in B-CLL: MHC Class II Restriction and Anti-Cytomegalovirus Reactivity.
Authors: Walton James , Lawson Keirissa , Manoussaka Maria S. , Nathwani Amit , Emory Vincent , Lydyard Peter M. , Porakishvili Nino
Published: April 2020

Introduction: An expansion of CD4+ T cells expressing perforin (PF) has recently been described in B-CLL and we have previously demonstrated anti-cytomegalovirus (CMV) reactivity within this population (Walton et al; 2004; Blood [ASH annual meeting abstracts] 104:4787). Here we further characterise the anti-CMV response of CD4+PF+ T cells in B-CLL and investigate the role of CMV in CD4+PF+ T cell expansion.

Methods: Peripheral blood mononuclear cells (PBMC’s) from 24 untreated B-CLL patients (17 CMV seropositive [SP], 7 CMV seronegative [SN]), 2 SP treated (Campath) patients and 12 healthy age-matched control individuals (8 SP, 4 SN) were fixed, permeabilised and stained with anti-CD4PerCP, anti-IFN-γ-APC and anti-PF-FITC monoclonal antibodies (mABs) (BD). PBMC were cultured for 18 hrs with DOWNE cell lysate (Dade Behring) containing CMV-antigen or lysate alone and with anti-CD28 and anti-CD49d mAbs (BD), in the presence of Brefeldin A (eBiosciences). In blocking experiments PBMC were pre-incubated with anti-HLA DR,DP,DQ mAb (BD) for 1 hour. The CMV specific response was assessed by flow cytometry (Dako Cyan, Summit software) as the percentage of IFN-γ+ cells in PF+ and PF− CD4+ T cell populations. Statistical analysis was performed using the Mann-Whitney U test and Spearman rank correlation.

Results: The proportion of CD4+ T cells expressing PF directly ex vivo was significantly higher in SP B-CLL patients (17.5±18.6%) compared to SN patients (2.0±2.3%, p=0.019). In seropositive aged matched controls the percentage of CD4+ cells expressing perforin was positively correlated with the percentage of CMV-reactive CD4+ cells (r=0.976, p<0.01). In contrast, there was no significant correlation in the patient group. However, two patients with relatively large expansions of CD4+PF+ cells (37.7±3.39%) post-Campath treatment had high percentages of CMV-reactive CD4+ cells (10.93±0.62%) compared to SP B-CLL patients (1.34±1.19%) and SP controls (1.31±1.14%), implying Campath related CMV reactivation. The addition of anti-HLA-DR,DP,DQ mAb to patients’ PBMCs, prior to CMV stimulation, led to an 80% (from 3.26% to 0.79%) and 90% (from 3.9% to 0.45%) reduction in the proportion of antigen reactive CD4+ and CD4+PF+ cells respectively.

Conclusions: A population of major histocompatibility complex (MHC) class II restricted, CMV reactive, CD4+PF+ T cells exists peripherally, in a large group of CMV SP B-CLL patients. Furthermore, CMV is associated with CD4+PF+ T cell expansion in patients and controls. Our data implies that high numbers of B-CLL cells inhibit anti-viral effector function, leading to increased viral activity and chronic antigenic exposure, potentially driving CD4+PF+ T cell expansion.
Sphingosine 1-Phosphate (S1P) Induces Migration and ERK/MAP-Kinase-Dependent Proliferation in Chronic Lymphocytic Leukemia (B-CLL) Due to Expression of the G Protein-Coupled Receptors S1P1/4.
Authors: Seitz Gabriele , Yildirim Sedat , Boehmler Andreas M. , Kanz Lothar , Möhle Robert
Published: April 2020

Egress of lymphocytes from lymphoid organs into the circulation has been shown to depend on the presence of the lipid mediator sphingosine 1-phosphate (S1P) in the peripheral blood, and expression of corresponding S1P receptors (i.e., S1P1), that belong to the family of 7-transmembrane G protein-coupled receptors (GPCR). As circulating lymphocytic lymphoma cells are a hallmark of chronic lymphocytic leukemia, we analyzed expression of different S1P receptors and the effects of S1P on B-CLL cells. By qualitative and quantitative (TaqMan) RT-PCR, significant mRNA expression of S1P1 and S1P4 was found in CLL cell lines (EHEB, MEC-1) and in most samples (S1P1 in 88%, S1P4 in 100%) of primary CD19+ cells isolated from the peripheral blood of untreated B-CLL patients. mRNA of other S1P receptors (S1P2, S1P3, S1P5) was less consistently detected. Normal, nonmalignant B cells were strongly positive for S1P1, while other S1P receptors were weakly expressed or negative. S1P induced typical effects of chemotactic GPCR, such as actin polymerization (analyzed by flow cytometry) and chemotaxis (measured in a modified Boyden chamber assay) in CLL cell lines and primary B-CLL cells. After serum deprivation in vitro, S1P induced phosphorylation of ERK/MAP-kinase as analyzed by Western blot, demonstrating that S1P receptors expressed in CLL were able to activate signaling pathways of GPCR not only related to cell migration and chemotaxis, but also to cell proliferation. Of note, the S1P1 ligand FTY720, which induces receptor internalization after prolonged exposure and acts as an antagonist, resulted in apoptosis in CLL cell lines and primary CLL cells in vitro, as measured by MTT-test and staining with Annexin-FITC, respectively. We conclude that sphingosine 1-phosphate, which is present in the peripheral blood in considerable amounts, contributes to the trafficking of B-CLL cells expressing the GPCRs S1P1/4, and to their prolonged survival.
CD38 Expression in Chronic Lymphocytic Leukemia (CLL) Patients Is Associated with Family History of Hematological Neoplasms.
Authors: Domingo-Domenech E. , Domingo-Claros A. , Gonzalez-Barca E. , Benavente Y. , Alvaro T. , Bellas C. , Font R. , de Sevilla A. Fernandez , de Sanjose S.
Published: April 2020

Introduction: Family clustering of hematological tumours has largely been reported in chronic lymphocytic leukemia (CLL) patients.

Objectives: To identify clinical and biological parameters at diagnosis that could be associated to family history of haematological neoplasms in CLL patients.

Methods: Eighty-five unselected consecutive CLL patients were included in the case-control multicenter study Epilymph_Spain. Immunophenotype data, including CD38 expression, genetics and clinical presentation were performed at diagnosis. Personnel interviews were conducted to collect data on family history of cancer, including site, age at diagnosis, and status of the affected relative. Statistical differences between each group were analysed using Chi-Square tests.

Results: Out of the 85 CLL patients included in the study, 50 (59%) reported a first-degree relative with cancer, from which 7 (8%) were haematological tumours. In the following table, the most relevant variables related with the family history of hematological neoplasms in CLL patients are presented.

Conclusions: Family history of haematological neoplasms, CD38 expression and younger age at presentation of the disease seems to identify a subgroup of CLL patients that could have a genetic origin.

Family history of Haematological neoplasm p NO (n=78) YES (n=7) Median age (range) 71 (45–87) 62 (53–74) 0,061 Gender (M/F) 51 /27 4 /3 0,476 CD38 + 21 (27%) 5 (71%) 0,026 Rai 0–1/2–4 67 /11 7 /0 0,369
Comparison of Genomic Aberrations and Expression of ZAP-70 and CD38.
Authors: Yañez Lucrecia , Cuadrado Maria Angeles , Insunza Andres , Bermudez Arancha , Mesones Belen Gonzalez , Conde Eulogio , Bureo Encarna , Iriondo Arturo
Published: April 2020

Expression of CD38 and /or ZAP 70 and chromosome abnormalities such as deletions of 11q23 and 17p13 have been previously identified by several groups as predictors of disease progression and decrease overall survival in Chronic Lymphocytic Leukemia (CLL). Moreover, few are described about the relationship between ZAP70/CD38 status and their genomic aberrations associated.

We analysed 86 patients (median age: 70 yrs; female/male: 28/58) diagnosed of CLL between 1980 and 2005. Cytoplasmic ZAP-70 expression and cell surface expression of CD38, was determined by flow cytometry in CD19+ CD5+ B-CLL cells. Trisomy 12 and deletions in 13q14, 11q22–23 and 17p13, were detected by the interphase cytogenetic fluorescence in situ hybridization (FISH). Biological study in patients diagnosed before 2001 was done on cryopreserved blood cells.

Concordant expression of ZAP-70 and CD38 was seen in 66 of 86 patients (76%). Fifty-one patients were ZAP-70- and CD38-, and 15 patients were ZAP-70+ and CD38+. Discordant ZAP70/CD38 status was present in 20 patients. Cytogenetic study identified chromosome aberrations in 85% of patients. Deletions in band 13q14 (43%), followed by trisomy 12 (21%), deletions in 11q22–23 (11%) and in 17p13 (10%) were the most frequent abnormalities. Normal karyotype was presented in 28% of patients and complex karyotype in 11%.

In the concordant ZAP70-CD38- cases and ZAP70+ CD38+, 13q14 aberration (47, 9%) and normal karyotype (42%) respectively, were more frequent than in the other groups but in our study this differences were not statistically significant. Moreover deletion in 11q22–23 (25%) was seen statistically significant, p = 0, 05, in patients with concordant expression of ZAP70+ and CD38+. Patients with discordant status in the expression of ZAP70 and CD38, were statistically associated with trisomy 12 (38%) p = 0,002.

In conclusion, in our study we find that expression in ZAP70 and CD38 is associated with a different genomic aberration. Deletion in 13q14 appears more frequent in patients with concordant ZAP70- and CD38- expression. Trisomy 12 is associated with discordant status expression. Finally, the worse prognostic group ZAP70+ and CD38+ is associated with a normal karyotype or deletions in 11q22–23.

Expression of ZAP 70 and CD38 and genomic aberrations ZAP70+ CD38+ ZAP70- CD38- Discordant status p-value Del 11q22-23 25% 4,2% 12,5% 0,05 Del 17p13 8,3% 4,2% ---- 0,52 Trisomy12 8,3% 10,4% 37,5% 0,002 Del 13q14 16,7% 47,9% 31,3% 0,10 Normal 41,7% 33,3% 18,8% 0,39 Complex Karyotype 20% 5,9% 20% 0,13
Inhibition of Bortezomib-Induced Apoptosis in Chronic Lymphocytic Leukemia by Red Blood Cell Uptake.
Authors: Wheat Luise M.C. , Kohlhaas Susan L. , Monbaliu Johan , De Coster Roland , Majid Aneela , Walewska Renata J. , Cohen Gerald M. , Dyer Martin J.S.
Published: April 2020

Bortezomib (PS-341/Velcade™) is a reversible inhibitor of the proteasome that has shown promising activity in clinical trials in several malignancies including multiple myeloma, mantle cell lymphoma and follicular lymphoma, including those with refractory disease. However, results have been less encouraging in chronic lymphocytic leukemia (CLL) and we have, therefore, sought to determine the barriers to effective therapy with bortezomib in this disease. Patients with CLL were eligible but were required to have received no therapy in the six months prior to the study. In a panel of 26 patients with CLL, both purified mononuclear cells and whole blood were tested for their apoptotic response to bortezomib (1–100 nM) up to 24 h by flow cytometry and western blotting. In all cases, purified CLL cells were sensitive to bortezomib-induced apoptosis in a concentration and time-dependent fashion, irrespective of stage of disease, resistance to prior therapy, IGHV mutational status or the presence of TP53 mutations. Apoptosis was induced at low (>10 nM) nanomolar concentrations of bortezomib by activation of the intrinsic apoptotic pathway. Bortezomib-induced apoptosis correlated with levels of ubiquitination, Bax activation, and caspase cleavage. Apoptosis of CLL cells was obtained at drug levels readily obtained in vivo using currently-used dosing protocols. However, in vitro, it was necessary to maintain these concentrations for 16–24 hours to obtain maximal apoptosis. Apoptosis measured in a whole blood apoptosis assay was markedly less than in isolated lymphocytes at comparable time points and concentrations. Activity of bortezomib in purified cells was not diminished by addition of exogenous plasma but was abrogated by addition of autologous red blood cells (RBC), suggesting preferential active uptake of the drug by these cells. These data were confirmed in animal models showing preferential distribution of bortezomib to the RBC fraction. RBC uptake may therefore account for the low serum levels of bortezomib attained in vivo during terminal half-life and thus the lack of activity against cells in the peripheral blood. Together with pharmacokinetic and in vivo data, these studies suggest that different dosing schedules of bortezomib other than bolus injections may be more effective in patients with CLL.
Xanthohumol Induces Apoptosis in B-CLL Mediated by Caspase 9 Following Downregulation of bcl-xL and mcl-1.
Authors: Vanhoecke Barbara , Lust Sofie , Janssens Ann , Philippe Jan , Bracke Marc , Offner Fritz
Published: April 2020

Introduction

Xanthohumol is a hop-derived prenylated chalcone with structural similarities to flavopiridol. It has documented impact on breast cancer cell growth and invasiveness in vitro. Based on these observations, lymphocytes from patients with 20 B-CLL were cultured in the presence of xanthohumol in vitro.

Methods

Xanthohumol was dissolved in DMSO and used at concentrations up to 50 μM. B-CLL cells were cultured in RPMI 1640–10% FBS in vitro. Apoptosis, cell cycle analysis and bcl-2 expression was assessed by flow cytometry. PARP, Erk1/2, p38, Akt, JNK, p70S6K, and P-p38, P-Akt, P-JNK, and P-p70S6K, caspase 3, 8 and 9, and bcl-XL and mcl-1 were assessed by Western Blot.

Results

After 24h, xanthohumol induced dose-dependent killing of B-CLL cells at a LD50 (24h) of 24.4 ± 6.6 μM, independently of known adverse prognostic factors such as Ig mutation status, ZAP-70, and cytogenetic abnormalities including 17p-loss of p53. Cell death was associated with PARP cleavage and Annexin V positivity, suggestive of an apoptotic mechanism. Apoptosis was accompanied by cleavage of procaspase-9 but not of procaspase-8. Among bcl-2 family members tested, there was a decrease in bcl-2, bcl-xL and mcl-1 expression. The effects of xanthohumol on the major signal transduction pathways showed no effect on Jnk, Akt, p38 and Erk phosphorylation, but stimulation of p70S6K phosphorylation which could not be inhibited by rapamycin, a specific inhibitor of mTOR.

Conclusion

Xanthohumol has antitumor activity on B-CLL cells in vitro that appears to be independent both of DNA-damage and of Zap-70 effects on signal transduction.
MDM2 Antagonists Activate p53 and Synergize with Genotoxic Drugs To Induce Apoptosis in B-Cell Chronic Lymphocytic Leukemia Cells but Not in T Cells.
Authors: Gil Joan , Coll-Mulet Llorenç , Iglesias-Serret Daniel , Santidrián Antonio F. , Castaño Esther , Campàs Clara , Barragán Montserrat , Cosialls Ana M. , De Frias Mercé , de Sevilla Alberto Fernández , Domingo Alicia , Vassilev Lyubomir T. , Pons Gabriel
Published: April 2020

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5+ B lymphocytes. Several drugs used currently in the therapy of B-CLL act, at least partially, through activation of the p53 pathway. Recently, non-genotoxic small-molecule activators of p53, the nutlins, have been identified that inhibit p53-MDM2 binding. We have investigated the antitumor potential of nutlin-3 in B-CLL and find that it can effectively activate the p53 pathway and induce apoptosis in cells with wild-type but not mutant p53. The half-maximal effective concentration (EC50) for B-CLL cells was 4.7 ± 1.5 μM (n = 8). Nutlin-3 stabilized p53 and induced several p53 target genes including MDM2, p21, PUMA, Bax, PIG3 and WIG1. At lower concentrations, nutlin-3 synergized with the genotoxic drugs doxorubicin, chlorambucil, and fludarabine but not with acadesine, that induces p53-independent apoptosis. Normal human T cells showed lower sensitivity to nutlin-3 than B-CLL cells and no synergism with the genotoxic drugs. These results suggest that MDM2 antagonists alone or in combination with chemotherapeutic drugs may offer a new treatment option for B-CLL.
The Research of Specific Inhibition of Survivin Expression To Reverse Drug Resistance with RNA Interference Technology.
Authors: Li Juan , Zhao Ying , Luo Shao-kai , Zhang Dian-bao , Haung Bei-hui
Published: April 2020

Objective:Use RNA interference technology to down-regulate the expression of survivin gene in KM3 cells, observe its induction of apoptosis of KM3 cells, and if it can increase chemotherapy sensitivity of KM3 cells to adriamycin.

Methods: SiRNA designed and in-vitro chemosynthesized aiming to survivin was transfected into human myeloma cell line KM3 mediated by liposome. Survivin mRNA transcription and protein expression of KM3 cells were then detected using RT-PCR and Western-blot 24, 48 and 72 hours after transfection, so as to invest the silencing effect. Besides, the apoptosis of the cell line were observed under fluorescence microscopy before and after transfection. Furthermore, cytotoxic analysis was used to compare the sensitivity variation of KM3 cells to adriamycin before and after transfection.

Results: Down-regulations of survivin mRNA transcription and protein expression of KM3 cells could be observed 24 hours after siRNA tranfection, which were (12±2.3)% and (9.4±1.8)% respectively. After 48 hours, they were (61.4±7.9)% and (38.6±6.7)% respectively, while the survivin mRNA transcription and protein expression was increased quickly 72 hours later. Under fluorescence microscopy, the apoptosis rate of KM3 cells transfected with survivin siRNA 48 hours later was 28±7%, which was significantly higher than the control cells. (P<0.05). The IC50 value of KM3 cells to adriamycin decreased from 1.12±0.14uM to 0.21±0.03uM, which means the sensitivity of KM3 cells to adriamycin was 5.3 fold increased.

Conclusions: SiRNA which was designed and synthesized to target survivin could effectively down-regulate survivin mRNA transcription and protein expression in KM3 cells, induce KM3 cells to apoptosis, increase its sensitivity to adriamycin after down-regulation, and effectively reverse drug-resistance of MM cells.
Validation of International Staging System (ISS) for Multiple Myeloma: A Retrospective Analysis of 487 Patients at 8 Brazilian Centers.
Authors: Hungria Vania T.M. , Maiolino Angelo , Martinez Gracia , Colleoni Gisele , Oliveira Luciana , Coelho Erika O.M. , Rocha Lais , Bittencourt Rosane , Quero Adriana , Ribeiro Karina
Published: April 2020

Introduction: The survival of patients with multiple myeloma varies from a few months to more than 10 years. This heterogeneity is related to the characteristics of the myeloma itself and of the host. The identification of the factors which influence the prognosis is very important to predict the result, assist in the choice of the treatment and adequately stratify the patients in clinical studies. Many prognostic factors have been identified in patients with multiple myeloma, such as anemia, renal failure, ß2 microglobulin, albumin and chromossomic alterations. Some authors have combined prognostic factors and proposed various systems of staging. However, none of them have yet substituted the Durie-Salmon staging system. Recently, the International Myeloma Working Group, with the objective of developing a simple and reliable staging system, which can be internationally applied to classify and stratify patients with multiple myeloma, identified 3 risk groups. This new system of staging, the “International Staging System” (ISS), consists of stage I: ß2 microglobulin < 3.5 mg/L plus albumin ≥ 3.5 g/dL (median survival: 62 months); stage II: neither I nor III (median 44 months); stage III: ß2 microglobulin > 5.5 mg/L (median 29 months). This study included sites in North America, Europe and Asia, but the sites in Latin America were not included.

Objective: To validate the ISS in patients with multiple myeloma at Brazilian centers.

Patients and Methods: Four hundred and eighty-seven patients with the diagnosis of multiple myeloma within the period of 1998 to 2004 at Santa Casa de São Paulo, Hospital Universitário Clementino Fraga Filho do Rio de Janeiro, Hospital das Clínicas de São Paulo, Hospital São Paulo, Hospital das Clínicas de Ribeirão Preto, HEMOPE, Hospital Prof. Edgar Santos de Salvador and Hospital de Clínicas de Porto Alegre, with available data on albumin and ß2 microglobulin, were stratified according to the ISS. A total of 339 patients received standard therapy and 148 received high-dose therapy as initial therapy. The survival was estimated using the Kaplan-Meier method with differences in survival examined using the logrank test.

Results: The median age of the patients was 60 years, 52% male and 48% female. In Stage I (n=104), the global median survival was not reached, the survival at 60 months was 60%, in stage II (n=264), the global median survival was 61 months and in stage III (n=119), 19 months (p<0.001).

Conclusion: The new system of staging for multiple myeloma (ISS) is simple, based on variables easy to be applied and was possible to be validated in patients with multiple myeloma in Brazilian centers.
Wnt3 Augments Adhesion Mediated Drug Resistance of Myeloma Cells Via Wnt/RhoA Pathway.
Authors: Chiba Hiroki , Kobune Masayoshi , Kato Kazunori , Nakamura Kiminori , Kawano Yutaka , Takimoto Rishu , Matsunaga Takuya , Kato Junji , Niitsu Yoshiro , Hamada Hirofumi
Published: April 2020

Despite the tremendous effort in developing of conventional chemotherapy and molecular targeting drugs for patients with multiple myeloma (MM), it has been proven difficult to completely abrogate neoplastic cells from bone marrow (BM). Hence, patients with refractory disease still experience poor outcome due to disease progression. Principle obstacle in the treatment of this disease is a chemo-resistance which is mainly caused by the interaction of myeloma cells with BM stromal cells. However, little is known about the molecular mechanism of cell adhesion mediated drug resistance (CAM-DR) in MM. In this study, we focused on relationship between drug resistance and expression of Wnts, the factor regulating the cell adhesion and proliferation, in myeloma cells. To gain insight into involvement of Wnt signaling in CAM-DR, we first screened the expression of Wnt family in myeloma cell lines (RPMI8226, ARH77, KMS-5, HS-sultan and MM1S) by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. Although the mRNAs of Wnt2b, Wnt7a and Wnt10b were variably expressed in some of myeloma cell lines, Wnt3 mRNA was detected in all the myeloma cells examined. RPMI8226, ARH77 and KMS-5 which highly expressed Wnt3 protein, tightly adhered to human BM stromal cells and accumulation of beta-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. This cell adhesion was augmented by addition of Wnt3 containing conditioned medium (CM) and suppressed by Wnt-receptor competitor, secreted Frizzled related protein (sFRP)-1, but not by specific inhibitor of canonical pathway (DKK-1). These results suggest that adhesion of myeloma cells was regulated by non-canonical pathway of Wnt signaling. We further examined whether the Wnt3 mediated adhesion to stromal cells involved in CAM-DR. The drug resistance of ARH-77 for doxorubicin was 1.8 folds enhanced by adhesion to stromal cells in comparison with stroma-free condition. This CAM-DR for doxorubicin was further augmented (2.6 folds) by addition of Wnt3 CM via enhancement of adhesion to stromal cells. Moreover, although the doxorubicin sensitivity of ARH-77 in coculture with stromal cells was significantly reduced by sFRP-1, this effect was not observed in stroma-free culture, indicating that Wnt3-mediated CAM-DR is dependent on attachment with stromal cells. Additionally, CAM-DR was completely restrained by addition of Rho kinase inhibitor Y27632. These results indicate that Wnt3 augments myeloma CAM-DR by enhancement of adhesion to human BM stromal cells via Wnt/RhoA signaling. Thus, Wnt/RhoA signaling pathway could be a promising molecular target to overcome CAM-DR.
Immunoglobulin Free Light Chains at Diagnosis: Predictors of Progression and Survival in Solitary Plasmacytoma of Bone.
Authors: Dingli David , Kyle Robert A. , Rajkumar Vincent S. , Nowakowski Grzegorz S. , Larson Dirk R. , Bida John P. , Gertz Morie A. , Dispenzieri Angela , Melton L. J. , Therneau Terry M. , Katzmann Jerry A.
Published: April 2020

Background: Solitary plasmacytoma of bone (SBP) is a localized collection of monoclonal plasma cells that is potentially curable with local radiation therapy but associated with a high risk of progression to multiple myeloma. We hypothesized that an abnormal immunoglobulin free light (FLC) ratio at diagnosis may be a prognostic indicator of transformation risk.

Methods: We identified a cohort of 133 patients with SBP for whom stored serum taken at the time of diagnosis was available. The diagnosis was ascertained and serum FLC determined in 126 patients.

Results: From this cohort, 48 patients have progressed to myeloma and the median time to progression among those who progressed was 1.9 years. On univariate analysis, age (p<0.001), gender (p=0.035), abnormal FLC ratio at diagnosis (p=0.009) and persistence of serum or urine M-protein after therapy (p=0.0070 were all associated with a shorter overall survival (OS) and time to progression to multiple myeloma.

Progression by Normal FLC(0.26–1.65) Progression by Normal FLC(0.26–1.65)

On multivariate analysis, an abnormal FLC ratio retained its independence in a model that includes age at diagnosis but lost its significance when combined with persistence of the serum or urine M-protein. However, serum or urine M-proteins are not detectable in a significant number of patients with SBP and therefore not informative.

Conclusion: The FLC ratio at the time of diagnosis of SBP is a powerful predictor of risk and a useful aid to management of patients with this condition.
Differential Presence of Truncated Forms of Transcription Factor Yin Yang 1 (YY1) in Bone Marrow of Multiple Myeloma Patients.
Authors: Neshat Mehran S. , Chen Haiming , Gordon Melinda S. , Berenson James R. , Bonavida Benjamin
Published: April 2020

The transcription factor Yin Yang 1 (YY1) regulates cellular differentiation and response to apoptotic stimuli. YY1 exerts its pleiotropic effects through regulation of promoter activity of critical genes, as well as association and direct modulation of stability and function of a subset of proteins. Genes that are regulated by YY1 include those that control the cell cycle, development, differentiation and tumor suppression. For example, it has been reported that YY1 inhibits the proto-oncoprotein c-Myc (Austen, et al., Oncogene, 1998, 17:511) and negatively regulates the tumor suppressor gene p53 (Sui, et al., 2004, Cell 117: 859). Thus, expression and activity of YY1 in tumor cells may be involved in the pathogenesis of disease, as well as controlling response to drug stimuli. YY1 is regulated at transcriptional and post-translational levels in response to intra and extracellular signals. It has been reported that YY1 undergoes proteolytic cleavage. Caspase-dependent N-terminal cleavage of YY1 has been reported in response to physiological (Fas, TNF, L-glutamate) and chemical (staurosporine, etoposide, okadaic acid) death promoting factors. Similar presence of truncated YY1 is observed in in vitro models of skeletal and cardiac muscle differentiation. N-terminal truncated YY1 lacks its transactivation domain, while DNA binding remains unaltered. Hence, YY1 function may be altered by truncated forms. We hypothesized that post-translational processing of YY1 occurs in bone marrow and may be important in tumor progression and response to therapeutic agents. This study thereby aimed to determine whether altered levels and/or forms of YY1 are expressed in the bone marrow of multiple myeloma patients and to identify their potential downstream effectors. YY1 expression in protein lysates of bone marrow aspirates from nine patients was determined by Western blot analysis. Truncated species of YY1 were present in 6/8 samples. In contrast to myeloma bone marrow, one plasma cell leukemia sample showed high levels of YY1 and no truncated forms. Similar high levels of YY1 expression was observed in established tumor xenografts of a plasma cell leukemia tumor. We are presently extending the pool of analyzed normal and cancer harboring tissues and examining potential correlation of YY1 and its altered forms with disease status and prior therapeutic history. Identification and purification of cell populations that generate altered forms of the protein and its effect on expression and function of YY1 interacting proteins are under investigation.
Circulating Nucleic Acids in Multiple Myeloma: Presence of Non-Coding/ Intergenic DNA Reflects Disease Activity.
Authors: Durie Brian G.M. , Jagannath Sundar , Schutz Ekkehard , Urnovitz Howard
Published: April 2020

Circulating nucleotides in plasma have been previously quantified and sequenced in studies involving 65 patients with multiple myeloma (Acta Onc39, 7892000). RT-PCR revealed RNA incorporating a 709-nucleotide sequence (GenBank #AFO18254) with 99.7% homology with a non-coding ALU sequence of a type capable of retro-transposition and disease linked mutation. Levels of this circulating ALU RNA correlated with myeloma disease status. Circulating DNA, devoid of cellular material was also noted as part of these studies. We now describe the molecular analysis of this circulating DNA before, during, and after therapy. Based upon prior findings, direct PCR was performed utilizing primers with a 3′ Alu sequence linked to a variety of 5′ sequences. Detailed studies including serial evaluations were conducted in one patient. 167 total clones were analyzed and categories set to compare under and over-representation in comparison of active myeloma samples versus samples from healthy controls. 96 selected clones fitted into 10 homologs. Homolog #2, which proved to be reflective of myeloma disease activity, incorporated only non-coding DNA regions. One homolog sequence, CNA Clone #2, matched one GenBank entry in the human genome (GenBank #AC020734; chromosome 4), incorporating only repetitive elements and interspersed DNA. The percentage occurrence rates for different types of repeat elements in CNA Clone 2 were: LINEs 25.2%; MaLR 5.5%; HERVs 4.8%; ALUs 4.8%; MERs 3.8%; MiR 2.1%; microsattelites 1.1% and A-T rich regions 1.0%. Using a combination of the 10 homologs containing ALU-attached sequences, distinct patterns of clonal change occurred over several months of follow-up in the patients studied. Initial clones associated with active disease disappeared with response. Transiently, expressed different clones emerged with intervening infection and disappeared with resolution of infection. Electron microscopy has revealed that circulating nucleotides are present in the form of exosomes or membrane vesicles released into the extra-cellular environment upon exocytic fusion of multivesicular endosomes with the cell surface membranes.

Conclusions: Our studies revealed several important aspects. Firstly, specific types of circulating DNA and RNA accompany the presence of active myeloma. Secondly, the predominant types of RNA and DNA are repetitive elements, especially SINEs, LINEs and ALUs. Thirdly, that these repetitive elements are typically linked to other non-coding sequences derived from interspace gene (intergenic) “hotspots”, which may be linked to particular patterns of disease and/ or treatment responses. The serial testing of DNA patterns has the potential for personalized molecular patient monitoring. Further studies are ongoing to assess these findings in a randomized trial setting and in comparison with proteomic and other markers.
Monitoring of Dendritic Cell Numbers in Patients during the Treatment of Multiple Myeloma.
Authors: Kovarova Lucie , Hajek Roman , Svobodnik Adam , Penka Miroslav , Mayer Jiri
Published: April 2020

Objective: In this study, the proportion of dendritic cell (DC) subsets (myeloid DC1 and plasmacytoid DC2), T cells, B cells and NK cells was evaluated in peripheral blood of patients with multiple myeloma (MM) before and during treatment including autologous transplantation. Also control group of healthy volunteers was evaluated.

Methods: Flow cytometric determination of relative cells number in unmanipulated peripheral blood was based on expression of the surface antigen: T cells (CD3/CD4/CD8), B cells (CD19/CD20), NK cells (CD3/CD16/CD56) and DC (CD83/HLA-DR/CD11c and CD83/HLA-DR/CD123).

Results: Significant difference (p<0.01) was found in initial values of CD83+ cells between the group of healthy volunteers (n = 15; mean count of CD83+ cells 0,26 ± 0,15%; ratio DC1/DC2 = 1,54) and the group of patients before treatment (n = 15; 0,15 ± 0,03% CD83+; DC1/DC2 = 4,55). After induction treatment with VAD regimen (vincristine, adriamycin, dexamethasone) in a group of patients was the mean percentage of DC higher (0,18 ± 0,04% CD83+ cells; DC1/DC2 = 4,77) than initial values. Administration of G-CSF again increased the total DC numbers (0,34 ± 0,11%; DC1/DC2 = 2,3) and intermediate levels of DC counts were found in the apheresis products (0,22 ± 0,05%; DC1/DC2 = 1,21). After engraftment there were found the highest relative DC numbers (0,50 ± 0,21%; DC1/DC2 = 1,85) in patients. Within six months after transplantation were achieved pretreatment DC values when compared with DC values of healthy volunteers (p<0,97) (0,24 ± 0,08%; DC1/DC2 = 1,57). Total numbers of T cells did not significantly differ during treatment only the reverse CD4/CD8 ratio was found in majority of patients within six month after the transplantation.

Conclusions: Untreated patients with MM have significant lower relative numbers of peripheral blood DC in comparison with healthy volunteers. The highest number of total DC was found after engraftment. The ratio DC1/DC2 showed relative majority of DC1 subtype and its the lowest value was found in the apheresis products. Normal DC values comparable with DC values of healthy volunteers were found in patients within six months after transplantation together with the reverse CD4/CD8 ratio.
The Anti-Myeloma Effect of Bortezomib Is Associated with Osteoblastic Activity.
Authors: Zangari Maurizio , Najarian Kevin B. , Esseltine Dixie L. , Lee Choon-Kee , Barlogie Bart , Elice Francesca , Burns Michael J. , Yaccoby Shmuel , Richardson Paul , Sonneveld Pieter
Published: April 2020

Myeloma bone disease is characterized by osteolytic destruction associated with suppressed osteoblastic activity possibly through inhibition of the WNT signaling pathway. We already reported the association between increased alkaline phosphatase (ALP) and B but not dexamethasone response in myeloma patients (Zangari et al., BJH 2005, in press). To extend these observations, using data from the large APEX study, we have attempted to assess the relationship between quantitative alkaline phosphatase (ALP) changes during therapy with B and response and time to progression. We were interested in what threshold of increase in ALP (even within the normal laboratory range) might predict efficacy.

Methods: A group of patients with no biochemical evidence of liver disease and creatinine clearances > 50 mL/minute treated with bortezomib 1.3 mg/m2 on days 1, 4, 8 and 11 for up to eight 3-week cycles followed by bortezomib 1.3 mg/m2 on days 1, 8, 15 and 22 for up to three 5-week cycles were studied. The percentage increase of ALP levels in responders and non-responders was analyzed at different thresholds and time points after baseline. Responses (the sum of CR plus PR) were based on EBMT criteria. The change from baseline ALP during therapy with B was compared in responders and non-responders.

Results: There were 229 patients with a median age of 60 years. 59% were male; median B2M was 3.2 mg/L, myeloma subtypes included IgG 64%, IgA 24%; median time from diagnosis was 41 months. The median ALP elevation from baseline for bortezomib responding patients was approximately 25% but even a 10% ALP increment at week 6 from baseline was strongly associated with response (P=0.0001). The most powerful predictor of response was a 25% ALP elevation at week 6.

Table 1 Parameter 25% ALP Elevation <25% ALP Elevation p-value N 64 154 CR n (%) 10 (16) 12 (8) 0.0966 PR n (%) 35 (55) 42 (27) <0.0001 CR/PR n (%) 45 (70) 54 (35) <0.0001

Cox regression analysis showed that ALP elevation of 25% was not only associated with response rate but was also positively correlated with time to progression (P=0.0022).

For all bortezomib treated patients enrolled in the trial (n=333) regardless of their baseline (LFTs and creatinine), a 25% increase in ALP from the baseline to 6 week remained strongly associated with a very high response rate (p<0.0001) and longer time to progression (206 vs 169 days) p=0.01.

Figure Figure

At the time of relapse after B therapy, the median ALP was 65.0 U/L and comparable to baseline level (65.5 U/L).

Conclusion:

During bortezomib treatment, markers of osteoblastic activation (such as ALP) may predict response and response duration in patients with multiple myeloma. In addition, this suggests that bone anabolism could affect myeloma growth. This observation needs to be confirmed prospectively.
Development of Retargeted CD38-Specific NK-92 Cell Line for Potential Anti-Myeloma Immunotherapy.
Authors: Yang Shihong , An Xin , Brown Ross D. , Ho Joy , Gibson John , Joshua Douglas E. , Wels Winfried , Sze Daniel M.
Published: April 2020

Multiple Myeloma (MM) is a B cell neoplasm in which malignant plasma cells accumulate in the bone marrow and produce excessive amounts of a monoclonal immunoglobulin. Among the large number of antigens expressed by plasma cells, CD38 is one of the highest expressed surface molecules, suggesting an alternative public tumour target. For instance, the myeloma cell line 8226 has a high surface expression of CD38. We plan to introduce genetically recombinant single-chain variable fragments (scFv) of anti-CD38 antibody, along with CD3 chain, into NK-92 a natural killer cell line which is highly cytotoxic against a variety of malignant cells (the only natural killer cell line being used in phase I/II clinical trials). It is likely that the retargeted NK-92 cells may specifically and efficiently kill CD38-expressing myeloma cells. By using a series of degenerate primer sets for mouse immunoglobulin variable region genes, VH and VL regions were isolated through reverse transcription PCR from OKT10 cell line. Further sequencing analysis provided novel information of the OKT10 immunoglobulin heavy and light chains. A PCR-based assembling strategy was employed to construct the scFv with FLAG sequence integrated as a tag. VH and VL domains were joined together with a hinge region of (Gly4Ser)3. The phagemid construct has been formed by subcloning the scFv fragment into the Sfi I/Not I cloning sites of the expression vector pHEN2, and subsequently transformed into the E. Coli HB2151. Five out of 7 transformed bacterial colonies screened did not contain the scFv fragment. For the remaining two clones with the correct-sized insert, we tested the specificity of the scFv fragments that were prepared through intracellular antibody capture as periplasmic proteins. We performed a competitive flow assay using periplasmic proteins with commercially available anti-CD38 PE conjugated antibodies on 8226 myeloma cell line. We found that one of these two clones (clone 6) showed a reduction in CD38 staining and presented as double peaks; while this pattern was not seen in the clone 7. Subsequent DNA sequencing revealed that there was a 100-bp truncation in the heavy chain region in clone 7; while the clone 6 has a complete productive heavy chain and light chain spanned by the linker region. We are going to transfect NK-92 cells with retrovial vector pL-scFv(clone 6) construct as the basis of an efficient approach for anti-myelome immunotherapy. (Dr. Daniel Sze is supported by the International Myeloma Foundation Senior Research Grant).
Chemosensitization of CDDP-Resistant Multiple Myeloma Cell Lines Following Treatment with Anti-TfR IgG3-Av.
Authors: Suzuki Eriko , Gan Xiao-Ho , Umezawa Kazuo , Helguera Gustavo , Schiller Gary , Penichet Manuel , Bonavida Benjamin
Published: April 2020

Multiple myeloma (MM) is an incurable proliferative disorder of monoclonal malignant plasma cells. Multiple agents conventional dose chemotherapy regimens have resulted in improved response rates, however, there have been no significant improvements in overall survival. Recently, Bortezomib has been introduced for the treatment of MM and has resulted in significant response rates. Nevertheless, additional strategies for direct treatment of MM patients are urgently needed. In general, cancer cells have been shown to express significantly higher levels of the transferrin receptor (TfR) and thus, this receptor is an attractive target for cancer therapy. We have generated an anti-TfR IgG3-avidin fusion protein (anti-TfR-IgG3-Av) that can deliver biotinylated molecules into cancer cells and exhibits intrinsic pro-apoptotic activity against hematopoietic malignant cells including myeloma (Ng P. et al., 2002, PNAS 99:10706–11). We have also reported that certain monoclonal antibodies (e.g., rituximab) can sensitize tumor cells to various chemotherapeutic drug-induced apoptosis (Jazirehi and Bonavida, 2005, Oncogene 65:264–76). Thus, this study investigated whether anti-TfR IgG3-Av sensitizes drug-resistant MM cell lines to drug-induced apoptosis. We initially examined the CDDP-resistant lymphoblastoid IM-9 and ARH-77 cell lines for chemosensitization using a suboptimal dose of anti-TfR IgG3-Av. Several methods were used to assess the cytotoxic activity, namely viable cell recovery and apoptosis by both PI and caspase-3 activation. The findings demonstrate that treatment of tumor cells with anti-TfR IgG3-Av for 18 h following treatment with CDDP for an additional 18 h resulted in significant cytotoxicity and apoptosis and synergy was achieved. The combined treatment also resulted in significant mitochondrial membrane depolarization, suggesting that apoptosis was achieved through a type-II pathway. In comparison with tumor cell treatment with anti-TfR-IgG3-Av, the anti-TfR IgG3 was a more potent chemosensitizer, based on both the concentration used and the extent of apoptosis achieved. These findings demonstrate that anti-TfRIgG3-Av is a potential therapeutic antibody that will reverse MM drug resistance when used in combination with chemotherapeutic drugs.