bluuebunny/crossref_metadata_2025 huggingface dataset:
c-kit D816V Provides a Strong Signal for Myelomastocytic Differentiation and Cluster Formation in Murine Ba/F3 Cells.
Authors: Mayerhofer Matthias , Aichberger Karl J. , Florian Stefan , Krauth Maria-Theresa , Bilban Martin , Esterbauer Harald , Wagner Oswald , Kodym Reinhard , Sotlar Karl , Longley Jack B. , Horny Hans-Peter , Sillaber Christian , Valent Peter
Published: April 2020

The pathologic hallmark of systemic mastocytosis (SM) is differentiation and cluster formation of mast cells (MC) in hematopoietic tissues. The somatic c-kit mutation D816V is detectable in a majority of all SM patients independent of the proliferation-status of MC or subtype (indolent or aggressive) of disease. To investigate the role of c-kit D816V in the pathogenesis of SM, we established a Ba/F3 cell line with doxycycline-inducible expression of c-kit D816V. We found that c-kit D816V provides a strong signal for mast cell differentiation and cluster formation in Ba/F3 hematopoietic progenitor cells without enhancing their growth thereby resembling the clinical presentation of indolent SM (ISM). As assessed by gene chip analysis, induction of c-kit D816V resulted in expression of various differentiation antigens including mouse mast cell protease 5, mi transcription factor, histidine decarboxylase (HDC), secretory granule proteoglycan, IL-4 receptor, ICAM-1, and CD63 consistent with an early phase of mastopoiesis. By contrast, c-kit D816V did neither induce expression of granulo-monocytic antigens such as myeloperoxidase, IL-3 receptor, or GM-CSF receptor, nor expression of ‘late stage’ mast cell antigens such as FcεRI. The c-kit D816V-induced synthesis of histamine in Ba/F3 cells was confirmed by RIA. To examine the role of c-kit D816V in the pathogenesis of mastocytosis, we extended our analysis to bone marrow biopsy sections obtained from patients with ISM. In these experiments, the D816V-mutated form of c-kit was detected more frequently in micro-dissected tryptase-positive MC obtained from dense compact infiltrates (44.2%) than in diffusely spread MC in these patients (22.6%) (p<0.05). In summary, our data establish a role for c-kit D816V in differentiation and cluster formation of neoplastic (mast) cells. Additional genetic hits, apart from c-kit D816V, may be responsible for aggressive growth of MC in advanced MC neoplasms.
Expression of the CD117 Antigen on Multiple Myeloma and Its Significance.
Authors: Li Juan , Luo Shaokai , Zhang Guocai , Hong Wende , Tong Xiuzhen
Published: April 2020

BACKGROUND & OBJECTIVE: Leukocyte differentiation antigen CD117 is one of the targets that tyrosine kinase selective inhibitors work on. CD117, whether the cell surface is expressed and the quality of its expression, is highly correlated with the tyrosine kinase selective inhibitors. And whether Multiple myeloma (MM) cells express CD117 and its expression quality is not reported domestic yet but only several reported overseas. In this study, CD117 expressed in MM cells is evaluated, which provide an theoretical evidence for tyrosine kinase selective inhibitors used in the MM, meanwhile, the value of the CD117 expressed in MM cells is estimated.

METHODS:CD117,CD56,CD54 were measured by three -color flow cytometry with CD45/SSC gating strategy.

RESULTS:Of 48 patients with MM, 17(35.5%) CD117 expression was positive in myeloma cells, and CD56, CD54 expression was positive in 39(81.2%), 48(100.0%), respectively. CD117 expression in myeloma cells was low compared with CD56, CD54 in 48 patients with MM; CD117 positive expression showed a positive correlation with myeloma cells in the bone marrow; CD117 positive in IgG type of MM was 64%, higher than other types such as light chain or IgA.CD117 positive showed no significant difference in different stage, untreated, relapsed and refractory patients (P>0.05, P>0.01); In untreated MM patient, chemotherapy of VAD in CD117 positive patient, the effectiveness was 71.4%, compared with the reaction rate 66.7% in CD117 negative, showed no significant difference (P>0.05).

CONCLUSIONS:CD117 </SU
The Proteomic Analysis of Sera for the Discovery of Biomarkers for Multiple Myeloma.
Authors: Kwak Jae-Yong , Lee Na-Ri , Song Eun-Gi , Yim Chang-Yeol , Kwak Yong-Geun
Published: April 2020

Multiple Myeloma (MM) fits in the group of plasm cell disorders characterized by neoplastic proliferation of single clone of plasma cell engaged in the production of a monoclonal immunoglobulin. The cause of MM is unclear, but a genetic predisposition may play a role and cytogenetic abnormalities are considered to be the most important prognostic factor. The proteomic analysis of MM emerges as a key complement to gene expression profiling, primarily because regulation of protein expression can buffer the magnitude of changes occurring at the gene transcription level. In the present study, to identify biomarkers of initial diagnosis, detection of relapse, monitoring for minimal residual disease and prognostic marker in MM by a lesser invasive method, serum proteins reflecting alteration in their proteomes were analyzed. We compared two-dimensional electrophoresis patterns of human sera of twelve patients with multiple myeloma with that of normal twelve subjects. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization-time-of flight (MALDI-TOF) and electrospray ionization quadupole time of flight (ESI-Q-TOF) mass spectrometry. We identified several proteins altered in sera of MM patients. These results suggest that these proteins can be used as a lesser invasive diagnostic, monitoring and prognositic biomarkers of MM if further studies were done.
Pre-Transplantation Level of Minimal Residual Disease Detected by Quantitative Real-Time IgH-PCR Is a Prognostic Parameter in Patients with Multiple Myeloma.
Authors: Fenk Roland , Korthals Mark , Kobbe Guido , Steidl Ulrich , Graef Thorsten , Rohr Ulrich-Peter , Ruf Leilani , Hoyer Barbara , Neumann Frank , Haas Rainer , Kronenwett Ralf
Published: April 2020

Background: High-dose chemotherapy with autologous stem cell transplantation has improved outcome and survival of patients with multiple myeloma. However, the majority of patients suffer from relapse. Using real-time quantitative (RQ) PCR we have shown before (Haematologica 89,2004) that the amount of residual tumor cells in the bone marrow of patients before transplantation is of prognostic relevance. In this study we evaluated in a larger group of patients with multiple myeloma whether a pre-transplantation level of clonotypic cells in the bone marrow is predictive for time-to-progression (TTP) and overall survival (OS). Further, we compared results with known prognostic factors.

Patients and Methods: Bone marrow samples of 19 patients with stage II/III multiple myeloma were obtained after induction therapy but before transplantation. Immunoglobulin heavy chain (IgH) RQ-PCR using patient-specific Taqman probes was performed to quantify pre-transplantation tumor levels. The proportion of clonotypic cells was assessed as IgH/2 beta-actin ratio in percent. Medical records of patients were reviewed for prognostic factors and outcome.

Results: The median level of residual tumor cells in bone marrow of all patients at the time before transplantation was 0.3 %. At 23 month median follow-up after transplantation the median TTP and OS in our study were 14 and 36 month, respectively. The threshold level of 0.03% clonotypic cells identified two prognostic groups (p<0.0001, log rank). Twelve patients in the bad prognostic group had an early relapse with a median TTP of 9 month (range: 3 – 17 month). All patients in the good prognostic group (n=7) had ongoing remissions after a median follow-up of 24 month (range: 13–44 month). Univariat analysis was performed including other prognostic factors at the time before transplantation such as cytogenetic abnormalities, beta2-microglobulin, hemoglobulin, platelet count, LDH, CRP, serum albumine and age. Besides the pre-transplant level of minimal residual disease, CRP level was predictive for TTP. In multivariat analysis using a step-wise cox regression model grouping by pre-transplantation tumor level was the only prognostic factor for TTP (p = 0.05). Moreover, low pre-transplantation tumor levels also showed a trend for a better OS, but in multivariat analysis only normal cytogenetics were predictive for a superior outcome (p = 0.03).

Conclusion: Quantitative molecular assessment of pre-transplantation tumor level in the bone marrow is an independent prognostic parameter for the progression-free survival of patients with multiple myeloma and thus helps to guide therapeutic interventions
Soluble Factors Secreted from CD40 Ligand-Transfected Dendritic Cells Enhance TRAIL-Induced Apoptosis of Multiple Myeloma.
Authors: Tomihara Kei , Kato Kazunori , Hamada Hirofumi
Published: April 2020

Dendritic cells (DCs) belong to a family of antigen presenting cells that play a crucial role in the regulation of cellular and humoral immune responses to bacteria, virus and cancer. Recent studies also revealed that IFN-stimulated monocyte-derived DCs expressed functional TNF-related apoptosis-inducing ligand (TRAIL), suggesting that DCs may become cytotoxic effector cells to tumors. In this study, we investigate whether gene transfer of CD40-ligand (CD40L), that is known to be a potent stimulator in DC maturation, could induce cytotoxic activity of DCs or modulate susceptibility to TRAIL that is expressed on cytotoxic T and NK cells. Human monocyte-derived DCs infected with adenovirus vector encoding human CD40L (CD40L-DC) were induced to express superior higher levels of T-cell costimulatory molecules, DC maturation markers, MHC molecules and the production of IL-12 than soluble CD40L or TNF-alpha treated DCs. In addition to the phenotypic alternation of CD40L-DC, we found that cultivation of multiple myeloma cells ARH-77 with CD40L-DC significantly inhibited the expression of phospho-AKT and induced apoptosis in vitro. However, we could not detect the expression of TRAIL and Fas-ligand on CD40L-DC by flow cytometry, suggesting that these molecules are not involved. To examine whether soluble factors produced by CD40L-DC contribute to tumor cell apotosis, we cultured myeloma cells MM1S or ARH-77 with CD40L-DC in transwell plates. We found that myeloma cells in transwell culture were not lysed for 48 hours, but the susceptibility of apoptosis by recombinant TRAIL protein could be increased. To investigate soluble factors that could affect TRAIL sensitivity of myeloma cells, we next tested the effect of neutralizing antibodies specific for CD40L and TRAIL or recombinant IFN-gamma that might modulate TRAIL sensitivity. However, antibodies and recombinant protein did not affect, indicating that these factors might be not involved. Our findings suggest that CD40L-transduced DCs have the ability of induction of TRAIL susceptibility of multiple myeloma cells mediated by soluble factors that are currently investigated.
The Chromosomal Pattern 14q-Translocation Plus 13q-Deletion Is Characteristic for Multiple Myeloma after a Preceding Monoclonal Gammopathy of Undetermined Significance.
Authors: Drach Johannes , Ackermann Jutta , Baldia Catrin , Kaufmann Hannes , Noesslinger Thomas , Gisslinger Heinz , Seidl Sonja , Sagaster Verena , Keck Andrea , Ludwig Heinz , Jaeger Ulrich , Zielinski Christoph
Published: April 2020

Multiple myeloma (MM) may be preceded by a monoclonal gammopathy of undetermined significance (MGUS), but it is at present unclear whether or not MM post-MGUS is biologically and clinically different from MM de-novo. To address this issue, we have performed a molecular cytogenetic analysis of 32 cases of MM post-MGUS (median time between recognition of MGUS and transition to MM, 7.6 years; range, 2.6 years to 19.5 years) and compared the findings with those of 256 patients with MM de-novo, in whom no previous history of MGUS had been documented. FISH studies of clonal plasma cells (cytoplasmic Ig positive) with probes for IgH translocations [t(14q32)], t(11;14)(q13;q32), t(4;14)(p16;q32), and deletion of 13q14 [del(13q14)] revealed results summarized in Table 1:

Serial studies of MGUS plasma cells and MM post-MGUS plasma cells from 12 of these patients have thus far indicated that all chromosomal abnormalities observed at MM post-MGUS were already present in the MGUS plasma cells; most notably, there was one patient with t(4;14) plus del(13q) who had both abnormalities at the time of MGUS 94 months prior to transition to MM. Collectively, our data suggest that MM post-MGUS is characterized by a distinct chromosomal pattern, in particular a high frequency of t(14q32) plus del(13q14), frequent occurrence of a t(11;14), but low frequency of a t(4;14). We are currently studying the t(14q32) plus del(13q) chromosomal pattern in MGUS to investigate its potential value as a risk factor for transition from MGUS to MM.

Table 1: FISH of MM post-MGUS versus MM de novo

Abnormality MM post-MGUS MM de-novo P-value Any t(14q32) 24/32 (75%) 114/256 (44.5%) .05 t(11;14)(q13;q32) 9/32 (28.1%) 32/256 (12.5%) .05 t(4;14)(p16;q32) 2/32 (6.3%) 27/256 (10.6%) .37 del(13q14) 19/32 (59.4%) 102/256 (39.8%) .13 del(13q14) plus t(14q32) 18/19 (94.7%) 57/102 (55.8%) .11 del(13q14) plus t(11;14) 6/19 (31.6%) 9/102 (8.8%) .03
High Levels of Soluble Immunoregulatory Receptors in Patients with WaldenströM’s Macroglobulinemia.
Authors: Hunter Zachary R. , Branagan Andrew R. , Santos Daniel Ditzel , Tournilhac Olivier , Hatjiharissi Evdoxia , Xu Lian , Manning Robert J. , Treon Steven P.
Published: April 2020

CD25, CD27, CD30 and CD40 are receptors for IL-2, CD70, CD153 and CD154, respectively, which provide important signaling for both B- and T-cell immune responses. We therefore examined the sera of patients with Waldenstrom’s macroglobulinemia (WM), a B-cell disorder characterized by excess IgM secreting bone marrow lymphoplasmacytic cells (LPC) for the presence of soluble CD25, CD27, CD30, CD40, IL-2, CD153, and CD154. Sera were from patients with active disease and off-therapy. Sera from healthy age matched donors (HD) was used for controls. The results of these studies were as follows:

WM ELISA Results HD ELISA Results p-value n= median range n= median range sCD25 pg/ml 2.647x10−6 41 3418.99 3–19756.2 20 573.6 184.96–891.03 IL-2 pg/ml 0.72385 41 22.96 3–76.83 20 11.07 5.64–64.7 sCD27 U/ml 2.4727x10−7 26 7.45 0–19.42 10 0 0–2.78 sCD70 ND ND ND ND ND ND ND sCD30 U/ml 0.00088 25 89.25 0–550.81 15 39.12 0–135.25 sCD153 ng/ml 0.68388 25 8.18 0–43.8 15 5.65 0–24.82 sCD40 pg/ml 0.02484 25 101.39 11.76–584.99 11 50.73 0–150.5 sCD154 pg/ml 0.81317 55 949.58 92–4973.16 17 1078.03 351.88–2323.38

Considering our recent studies demonstrating a role for mast cells (MC) in supporting WM cell growth (JCO 2004 22:571S), we examined bone marrow LPC along with MC from WM patients for expression of CD25, CD27 and CD40 and/or their ligands by flow cytometry and RT-PCR analysis. The results of these studies are as follows:

Flow Cytometry RT-PCR WM MC WM MC CD25 4/10 (40%) 2/7 (29%) 7/9 (78%) 7/15 (47%) IL-2 ND ND ND ND CD27 5/12 (42%) 2/8 (25%) 7/7 (100%) 4/7 (57%) CD70 6/6 (100%) 10/11 (91%) 7/7 (100%) 2/7 (29%) CD30 1/21 (4.7%) ND ND ND CD153 3/7 (43%) 12/13 (92%) 2/2 (100%) 9/11 (82%) CD40 25/30 (83%) ND ND ND CD154 2/3 (67%) 9/13 (69%) 7/9 (78%) ND

The above studies demonstrate high levels of circulating immunoregulatory receptors, and expression of these receptors and/or their ligands on WM tumor and mast cells. Studies addressing their role in immune dysregulation in WM are underway.
CD52 Is Expressed on Human Mast Cells and Is a Therapeutic Target for the Anti-CD52 Monoclonal Antibody Campath-1H in Waldenstrom’s Macroglobulinemia and Mast Cell Disorders.
Authors: Santos Daniel Ditzel , Hatjiarissi Evdoxia , Tournilhac Olivier , Xu Lian , Branagan Andrew , Hunter Zachary , Catsells Mayan , Anderson Kenneth C. , Treon Steven P.
Published: April 2020

Campath-1H is a monoclonal antibody used in the treatment of CD52 expressing B-cell malignancies. Recently, we and others have observed that Campath-1H shows unusually high activity in patients with relapsed/refractory Waldenstrom’s macroglobulinemia (WM), a B-cell malignancy with excess bone marrow mast cells (BMMC). Importantly, as we have recently shown, BMMC appear to provide direct support for WM tumor cell growth (Tournilhac et al, JCO 2004 22:571S), and are therefore a potential therapeutic target in WM. We therefore examined BMMC from patients with WM and other mast cell disorders for cell surface expression of CD52, and targeting by Campath-1H in preclinical studies. Multicolor flow cytometric analysis demonstrated CD52 expression on BMMC (FceRI+, CD117+) from 13/15 WM; 2/2 systemic mastocytosis (SM) patients; 2/4 healthy donors; as well as on the LAD and HMC MC lines. Moreover, RT-PCR analysis confirmed CD52 expression in sorted BMMC from 6/7 WM, along with 6/6 healthy donors. Importantly, Campath-1H induced high levels of antibody dependent cell mediated cytotoxicity (ADCC) actvity against LAD mast cells using activated NK effector cells. No direct cytotoxicity or antiproliferative activity by Campath-1H on LAD cells was observed. These studies demonstrate that CD52 is widely expressed on human MC and provide support for the use of Campath-1H in the treatment of WM and other systemic mast cell disorders.
IgA and IgG Hypogammaglobulinemia Persists in Most Patients with Waldenstrom’s Macroglobulinemia Despite Therapeutic Responses, Including Complete Remissions.
Authors: Treon Steven , Branagan Andrew , Hunter Zachary , Santos Daniel Ditzel , Tournilhac Olivier , Hatjiharissi Evdoxia , Xu Lian , Manning Robert
Published: April 2020

Recurrent infections are common in pts with Waldenstrom’s macroglobulinemia (WM) and may be related to IgA and IgG hypogammaglobulinemia. The impact of therapy and therapeutic responses on humoral immunity in WM is therefore of clinical interest. We examined IgA and IgG levels before and after therapy in 92 serially treated and responding WM patients. Their median age was 59 yrs and 70/92 (76.1%) were previously untreated. Median follow-up for all patients was 11 (range 3-134) months. Pre-therapy, 72/92 (78.2%) and 70/92 (76.1%), and post-therapy 82/92 (89.1%) and 80/92 (86.9%) pts demonstrated IgG (<700 mg/dL) and IgA (<70 mg/dL) hypogammaglobulinemia, respectively. Changes in median IgG and IgA levels by therapy were as follows:

N= Pre-IgG Post-IgG T-Test Pre-IgA Post-IgA T-Test Nucleoside analogue 5 438 399 0.63 32 38 0.35 Rituximab 17 455 358 0.29 35 29 0.43 Rituximab/Thalidomide 19 388 374 0.3 28 24 0.32 Rituximab/Nucleoside 21 571 423 0.006 55 29 0.05 CHOP 3 702 568 0.62 48 30 0.82 CHOP/Rituximab 14 537 383 0.1 61 36 0.21 Chlorambucil ± steroids 5 837 659 0.57 129 66 0.52 Campath-1H 7 521 444 0.62 28 27 0.61 HD Dexamethasone 1 269 173 NA 27 28 NA All therapies 92 480 398 0.008 38 28 0.01

Changes in median IgG and IgA levels by response category were as follows:

N= Pre-IgG Post-IgG T-Test Pre-IgA Post-IgA T-Test CR 8 644 472 0.05 75 37 0.08 PR 70 415 349 0.09 27 28 0.91 MR 14 474 394 0.01 38 26 0.02

These studies demonstrate that IgA and IgG hypogammaglobulinemia persists despite therapeutic responses, including complete remissions in patients with WM. Futhermore, the therapeutic regimen employed may aggravate persistent IgA and IgG hypogammaglobulinemia.
Preliminary Report of Risk-Based Approach in Korean Patients with Newly Diagnosed Multiple Myeloma.
Authors: Bang Soo-Mee , Lee Jae Hoon , Yoon Sung-Soo , Park Seonyang , Lee Je-Jung , Kim Hyeoung Joon , Jo Doeg Yeon , Park Yeon Hee , Yoon Hwi-Joong , Yuh Young-Jin , Ahn Myung-Ju , Im Seock Ah , Lee Moon Hee , Lee Dong Soon
Published: April 2020

Purpose: The purpose of this study was to examine the impact of different approaches stratified on risk based on chromosome 13 deletion and serum beta-2 microglobulin (MG) level would lead to survival benefit in patients with newly-diagnosed multiple myeloma.

Patients and Methods: At diagnosis, fresh marrow samples for FISH and serum for beta-2 MG were sent to central laboratory and reviewed. Patients who had chromosome 13 deletion and high beta-2 MG (>2.5 mg/L) were considered to have high-risk disease. Patients without chromosome 13 deletion and low beta-2 MG were classified as low-risk group. Intermediate-risk group had to have either one of these two risk factors. After VAD induction chemotherapy, autologous stem cell transplantation conditioned with MEL200 was performed in patients at high- and intermediate-risk. DECP consolidation chemotherapy was added for high-risk patients. Patients who achieved CR after VAD in low-risk did not receive any further treatment.

Results: As of Jun 2004, 50 patients were registered from 10 centers. Median age was 58 (range, 39–68) years old. Chromosome 13 deletion was detected in 18 patients (36%) and beta-2 MG was elevated in 39 patients (78%). Thirteen patients were classified as high-risk, 31 patients as intermediate-risk and 6 patients as low-risk. After median follow-up of 9 months, progression-free and overall survival at 1-year were 56% and 76%, respectively. To date, no statistically significant differences in survival were observed between risk groups (figure 1).

Conclusion: In this study, risk-based approach in patients with multiple myeloma appeared to be feasible. Study accrual is ongoing and updated results will be presented.

Figure Figure
Protective Effect of VELCADE® on Thalidomide-Associated Deep Vein Thrombosis (DVT).
Authors: Zangari Maurizio , Barlogie Bart , Lee Choon-Kee , Tricot Annick E. , Fassas Athanasios , Anaissie Elias , Hollmig Klaus , Thertulien Raymond , Talamo Giampaolo , van Rhee Frits , Stover Julie M. , Fink Louis , Tricot Guido J.
Published: April 2020

Cardiovascular complications during the treatment of patients with multiple myeloma (MM) are not uncommon (10%) and the frequency clearly increases with the use of regimens containing thalidomide (T) in combination with glucocorticosteroids or chemotherapy especially adriamycin. Even with prophylactic anticoagulation, DVT still occurs in 10% of such patients. The use of full anticoagulation treatment raises considerable concern of bleeding, especially during the post chemotherapy thrombocytopenic period. We now report the incidence of DVT in MM patients treated with T containing regimens where VELCADE has been added to dexamethasone (D) and adriamycin. Results are shown in table1.

The incidence of DVT was also analyzed in 24 patients enrolled on protocol UARK 2001–37, where VELCADE was administered in combination with thalidomide and dexamethasone (VTD) for a total of 98 cycles without anticoagulation. Review of the literature regarding the incidence of DVT in patients receiving DT reveals reports of 16% (Cavo, et al. Haematologica. 2004 89:826) and 12 % (Rajkumar, et al. JClinOncol. 2002 1;20: 4319). In contrast no thrombotic episodes were documented with any of these cycles. This is the first report of protective antithrombotic effect of VELCADE in a malignancy associated with a hyper-coaguable state. Further studies to elucidate the mechanism of VELCADE anticoagulant activity are currently in progress.

Table 1. DVT Rates: DTPACE vs. VDTPACE

Protocol Treatment N Pts reporting DVT (%) P=0.0006 UARK 2001–12 (DTPACE) Dexamethasone Thalidomide Cisplatin Adriamycin® Cyclophosphamide Etoposide Lovenox® (40mg sc qd) 98 10 UARK 2003–33 (VDTPACE) Velcade® (1mg/m2) Dexamethasone Thalidomide Cisplatin Adriamycin® Cyclophosphamide Etoposide Lovenox® (40mg sc qd) 69 0
Expression of Heat Shock Protein 90 and the Effects of HSP90 Inhibitor (17-AAG) in Multiple Myeloma.
Authors: Alkan Serhan , Duus Jan , Bahar Ismail H. , Izban Keith F. , Al-Masri Hytham , Maududi Tazeen , Toor Amir
Published: April 2020

Heat Shock Protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated antitumor effect in solid tumors. In order to test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analyzed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55 % also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL and MCL-1. Furthermore, while low concentration of bortezomib had no cell death, combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.
Anti-Tumor Activity of IPI-504, a Novel Hsp90 Inhibitor in Multiple Myeloma.
Authors: Palombella Vito J. , Normant Emmanuel , Ali Janid , Barrett John , Foley Michael , Gao Yun , Ge Jennie , Georges Asimina T. , Grayzel David , Grenier Louis , Hudak Jebecka , Pak Roger , Patterson Jon , Pien Christine , Pink Melissa , Porter James , Sang Junbiao , Spear Kerry , Sydor Jens , Wright Jim , Mitsiades Constantine S. , Anderson Kenneth C. , Adams Julian , Tong Jeffrey
Published: April 2020

IPI-504 is a novel inhibitor of Hsp90 based on the geldanamycin pharmacophore. When placed in rat, monkey, and human blood, IPI-504 rapidly converts to the known and well-studied compound 17-allylamino-17-demethoxy-geldanamycin (17-AAG). 17-AAG is the subject of multiple clinical trials for the treatment of hematologic and solid tumors. However, 17-AAG suffers from poor aqueous solubility necessitating the use of sub-optimal formulations to deliver this agent to patients. IPI-504 is over 1000-fold more soluble than 17-AAG in aqueous solution. In vitro, both 17-AAG and IPI-504 bind tightly to, and selectively inhibit Hsp90 derived from cancer cells. The cytotoxic effect of IPI-504, as well as its ability to stimulate the degradation of Hsp90 client proteins and increase the intracellular levels Hsp70, were monitored in two human multiple myeloma cells lines (RPMI-8226 and MM1.S). The effects of IPI-504 were compared to 17-AAG. We demonstrate that the actions of IPI-504 are bioequivalent to 17-AAG and that both compounds induce apoptosis in these cells and stimulate the degradation of HER2 and c-Raf. In addition, both agents stimulate Hsp70 protein levels. In all cases the EC50s are virtually the same for both molecules (~200–400 nM). Furthermore, IPI-504 inhibits the secretion of immunoglobulin light chain from the RPMI-8226 multiple myeloma cells (EC50 ~300 nM). Importantly, IPI-504 is active in tumor xenograft models of multiple myeloma. The data indicate that active metabolites of IPI-504 accumulate in these xenografts long after these metabolites are cleared from the plasma compartment, suggesting that they preferentially accumulate in tumor cells based on their increased affinity to Hsp90 derived from tumor cells. In conclusion, we have developed IPI-504 as a novel, potent inhibitor of Hsp90 with greatly increased solubility over 17-AAG, and that IPI-504 is an active anti-tumor agent in vitro and in vivo.
CHOP Plus Rituximab (CHOP-R) in Waldenström’s Macroglobulinemia (WM).
Authors: Hunter Zachary , Branagan Andrew , Treon Steven
Published: April 2020

Recently, a consensus panel of experts recommended alkylator therapy, nucleoside analogues and rituximab as appropriate first line therapies for WM (Semin Oncol2003; 30:121). It was also recommended that candidates for future autologous transplantation should have limited alkylator or nucleoside analogue exposure due to potential stem cell harm. CHOP-R (cyclophosphamide, adriamycin, vincristine, prednisone, rituximab) is a highly active, stem cell sparing regimen that has been extensively evaluated in other low-grade Non-Hodgkin’s lymphoma patients. No published experience in WM exists. As such, we analyzed the outcome of 13 patients with WM who received CHOP-R at our Institution. Patients had a median age of 54 (range 44–72) yrs, and a median of 1 (range 0–5) prior therapies. 8 of 13 (62%) patients had relapsed (n=2) or were refractory (n=6) to fludarabine. Five patients had received rituximab previously. Patients received a median of 6 cycles (range 2–6) of standard dose CHOP, and 6 infusions (range 2–6) of Rituximab (375 mg/m2). Three patients received additional rituximab (4 weekly infusions every 6 months) as maintenance therapy. Median IgM for all patients declined from 4,975 (range 1,960–12,400) to 1,575 (range 62–8,230) mg/dL (p=7 x 10−5); median S.V. 2.8 (range 1.5–12) declined to 1.6 (range 1.3–4.8) CP (p=0.04). Importantly, the median hematocrit rose from 30.7 (range 21.9–35.6) to 39 (range 24.3–43.2) p=0.005. Clinical responses were as follows: 3 CR/CRu; 7 PR; 2 MR. One patient is in stable disease after 3 cycles of CHOP-R. With a median follow-up of 8 (range 3–28) months, no patient has progressed. Therapy was well tolerated for most patients. Two patients had febrile neutropenia with documented bacteremia and recovered without any complication. Circulating effector cell levels pre- and post-CHOP-R were also evaluated in 6 patients since rituximab activity is mediated in part by ADCC activity. No significant change in CD3+, CD4+, CD8+, and CD16+/CD56+ effector cell levels occurred following CHOP-R as assessed by multicolor flow cytometry.
Metaphase Cytogenetic Abnormalities (M-CA) and Interphase FISH for Deletion 13 (FISH 13) in Total Therapy 2 (TT 2): Follow up Observation among? 380 Patients with Newly Diagnosed Multiple Myeloma (MM).
Authors: Shaughnessy John , Zhan Frank , Barlogie Bart , Rasmussen Erik , Milner Teresa , Jackson Lisa , Fassas Athanasios , Zangari Maurizio , Lee Choon-Kee , Talamo Giampaolo , Thertulien Raymond , Crowley John , Tricot Guido
Published: April 2020

We have previously reported on the adverse prognostic implications m-CA, present in ~ 33% of newly diagnosed patients especially when involving del 13 (~ 20%). To overcome the proliferation dependency of m-CA, FISH has been applied detecting abnormalities in virtually all patients with MM; FISH 13, detected in ~ 50% has been associated with poor outcome with both standard and high dose therapies. Of a total of 668 patients accrued to Total Therapy 2, 380 had baseline evaluations for m-CA and FISH 13. We examined 2 different cut-points of FISH 13 deletion, the traditional ≥ 20% (FISH 13–20) and an 80% cut-off (FISH 13–80). In case of FISH 13–20, major differences in both EFS and OS were noted among those exhibiting CA, with an adverse effect of FISH 13–20, whereas there was no difference among the majority of patients exhibiting no CA (Figure 1). However, using FISH 13–80, adverse effects of FISH 13 were observed for both no CA and CA subgroups (FISH 2). Indeed, 4-yr estimates of EFS/OS were highest at 73%/85% among 204 patients in the no CA/FISH 13–80-negative group compared to 54%/69% among the 42 with no CA and FISH 13–80-positive group. Similarly, among 134 patients with CA, those with FISH 13–80-positive disease had 4-yr EFS/OS of only 23%/22% compared 44%/58% among the 103 without FISH 13–80. Our data confirm the overriding importance of m-CA for prognosis and, in addition, reveal the importance of the proportion of plasma cells revealing del 13 as an additional key parameter in both no CA and CA subgroups. Biologically, these data would suggest a marked clinically relevant heterogeneity of FISH 13 that deserves further evaluation.
Plasmacytoid Precursor Dendritic Cells Contribute to the Ability of CD8+TCR− Facilitating Cells Population of Enhance Hematopoetic Stem Cell Engraftment and HSC Clonogenicity.
Authors: Fugier Isabelle J. , Rezzoug Francine , Huang Yiming , Schanie Carrie L. , Ildstad Suzanne T.
Published: April 2020

The discovery of cells with facilitative property offers great therapeutic potential in hematopoietic stem cell (HSC) transplantation. CD8+/TCR− facilitating cells (FC) enhance engraftment of HSC in allogeneic recipients without causing GVHD and also significantly enhance the engraftment of limiting numbers of HSC in syngeneic recipients. We recently identified that plasmacytoid precursor dendritic cells (p-preDC), the main component of the heterogeneous FC population, contribute to facilitation, but with less efficiency than the total FC population. In the present study we determined the role p-preDC play in the ability of FC to 1) enhance syngeneic suboptimal HSC engraftment and 2) maintain HSC clonogenicity in vitro. 500 syngeneic HSC +/− 30,000 FC, or p-preDC from the FC (DC-FC) were transplanted into ablated (950 cGy) B6 mice. As expected, FC significantly increased HSC engraftment, with 87.5 % of recipients (n=19) surviving at 250 days after transplantation, while recipients of HSC alone exhibited significantly impaired survival (37.5%, P=0.0026, n=16) (Figure 1). However, DC-FC, which allowed a slight improvement of HSC engraftment (52.6% recipient survival, n=16, P=0.15), were significantly (P=0.034) less effective than total FC. These data show that DC-FC are less effective than FC total. We then evaluated the influence of p-preDC and DC-FC on HSC in vitro using the colony forming cell (CFC) assay that enumerates progenitors in the HSC population. HSC were cultured in the presence or absence of FC, p-preDC or DC-FC (at 1:3 ratio) in methylcellulose containing growth factors for 14 days. The number of CFC was significantly increased when HSC were cultured in the presence of FC (P<0.01) compared to HSC alone (Figure 2). In contrast, the DC-FC slightly increased clonogenicity, as did p-pre-DC, but did not replace FC total. Moreover, when we preincubated HSC with FC, DC-FC or p-pre-DC for 18 hour and assessed in the CFC assay, FC significantly increased the number of CFC although no significant effect was observed with DC-FC or p-pre-DC (Figure 2). These data suggest that the effect of FC on HSC clonogenicity and HSC engraftment is only partially due to the contribution of the DC subpopulation, and that this effect on HSC requires synergistic interactions between the cells within the FC population. Moreover these data also suggest that FC facilitation on HSC involves a combination of different mechanisms: one on host alloreactivity due likely to DC-FC and one on direct interaction with HSC due to combined effects of cells in the FC.

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NF-κB Inhibitor Pyrrolidinedithiocarbamate Can Increase the Infection and Activation of Cytomegalovirus in Mesenchymal Stem Cells.
Authors: Wei Guoqing , Lin Maofang , Huang He , Cai Zhen
Published: April 2020

Objective Because of the central role of the transcription factor nuclear factor-κB (NF-κB) in cell survival and proliferation in many kinds of cancer cells, NF-κB inhibitor may have a potential role in the therapy of cancer. Human cytomegalovirus (CMV) infection is one of the most common complications following stem cells transplantation. Some data report that CMV infection has a very close relation with NF-κB activation. But it is unknown what effect of NF-κB inhibitor pyrrolidinedithiocarbamate (PTDC) on the infection and activation of cytomegalovirus in mesenchymal stem cells(MSCs).

Methods MSCs were infected by 1 TCID50 of CMV combined with/without 1μmol/L of PTDC. After 48h of culture with DMEM supplemented with 10% (v/v) fetal calf serum, MSCs shapes were observed. RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene and GAPDH gene. Flow Cytometry was used to detect the CMV PP65 antigen positive cells.

Results Shape of some MSCs changed after the infection of 1 TCID50 of CMV. But in MSCs infected by 1 TCID50 of CMV combined with 1μmol/L of PTDC, Cell shape changed more dominantly, almost all cells changed from thin shuttle shape to round and thick ball shape. While in the cells treated only with PDTC, shape of the cells did not changed. RT-PCR assay showed that there was a very bright band of CMV IE mRNA in MSCs infected by 1 TCID50 of CMV combined with 1μmol/L of PTDC compared with the cells infected only with 1 TCID50 of CMV. More CMV pp65 antigen positive cells were found in MSCs infected by 1 TCID50 of CMV combined with 1μmol/L of PTDC with Flow Cytometry.

Conclusion NF-κB activation may affect CMV infection of cells. NF-κB inhibitor PTDC can increase the infection and activation of CMV in MSCs and we should pay more attention to CMV infection when we use NF-κB inhibitor in clinical work..
Co-Transplantation of Hematopoietic Stem Cells and GM-CSF/G-CSF/CSF-Transfected Mesenchymal Stem Cells in SCID Mice.
Authors: Han Jin-Yeong , Koh Rhee-Young , Seo Su-Yeong , Park Joo-In , Kwon Hyuk-Chan , Kim Jae-Seok , Lee Young-Ho , Kim Hyo-Jin
Published: April 2020

Mesenchymal stem cells (MSC) are multipotent and believed to facilitate the engraftment of hematopoietic stem cells (HSC) when transplanted simultaneously in animal studies and recently even in human trials. In this study, we transfected culture-expanded MSC with GM-CSF, G-CSF, and CSF cytokine genes and then co-transplanted with HSC to further promote HSC engraftment. Mononuclear cells were harvested from the various sources and seeded in long-term culture for ex vivo MSC expansion. The phenotype and purity of MSC were assessed by flow cytometry. We transferred the above three cytokine genes into ex vivo expanded MSC, confirmed transfection by fluorescent microscope of GFP, and thereafter did co-transplantation with HSC. A total of 1x107 HSC plus MSC/uL were introduced to tail vein of SCID mice. After 3–7 weeks later, with venous blood from the eyeballs, homing and engraftment of human cells were determined by flow cytometry and fluorescence in situ hybridization (FISH) studies. The total nucleated cell count and the engraftment of CD45+/CD34+ cells and XX/XY-positive human cells significantly increased in co-transplanted mice and even higher with the cytokine gene-transfected MSC in the order of GM-CSF, SCF, and G-CSF transfections (P<0.05). These results suggest that MSC transfected with hematopoietic growth factor genes are capable of enhancing the hematopoietic engraftment. Now we are planning to deliver genes involved in homing and cell adhesions, e.g., CXCR4, VLA, or TPO into ex vivo expanded MSC and do co-transplantation with HSC to further increase the efficiency of stem cell transplantation.
An Exaggerated B Cell CpG Response in Human Chronic Graft-versus-Host Disease: A Potential Mechanism and Biomarker for Diagnosis of Chronic GVHD.
Authors: She Kevin , Gilman Andrew L. , Schultz Kirk R.
Published: April 2020

Chronic Graft-versus-Host Disease (GVHD) is a major limitation to successful allogeneic bone marrow transplantation (BMT). Lipopolysaccharide (LPS), a cell-wall component of gram negative bacteria that signals through Toll-like receptor 4 (TLR4), is an established contributor to acute GVHD. Our lab has previously shown that splenocytes from mice with acute GVHD were hyper-responsive to CpG, unmethylated DNA with C-G repeats from bacteria and viruses, which induce rapid responses by dendritic cells, B cells, and monocytes through TLR9. As part of Children’s Oncology Group phase III randomized trial ASCT0031 evaluating the efficacy of hydroxychloroquine in the treatment of newly diagnosed chronic GVHD, peripheral blood was drawn for immune phenotype and functional studies in patients and control (no GVHD, 80–120 days post allogeneic transplant). The non-BMT group were healthy adult volunteers. After Ficoll isolation, lymphocytes were cultured with or without synthetic human immunostimulatory CpG 2006 (6μg/mL) or LPS (1μg/mL) for 48 hours and analyzed by flow cytometry. Cell surface marker expression was evaluated as fold difference between stimulated and unstimulated cells. In some samples, B cells were purified by negative selection to determine proliferative response and/or for the quantitation of TLR9 mRNA. Chronic GVHD samples had a 3.7 +/− 1.2 fold (n=11) increase in B cell CD40 mean channel fluorescence after CpG stimulation which was significantly greater than in the control group at 1.0 +/− 0.6 fold (n=3, Mann-Whitney U test p=0.02) and the non-BMT group at 1.5 +/− 0.9 fold (n=3, p=0.01). Similarly, chronic GVHD samples had a 9.1 +/− 5.7 fold (n=10) increase in the percentage of B cells expressing CD86 after CpG stimulation which was significantly greater than the control group at 2.1 +/− 2.1 fold (n=4, p=0.008) and the non-BMT group at 2.7 +/− 0.9 fold (n=5, p=0.008). Responses to LPS stimulation or culture without stimulation were not significantly different between the three groups. Purified B cells from GVHD samples (n=3) had enhanced mitogenic response to CpG compared to the control group (n=1), non-BMT group (n=2), and to non-stimulated cells. CpG response appears to be closely correlated with TLR9 mRNA expression (R2=0.82). These findings suggest that CpG responsiveness or TLR9 mRNA expression may potentially be useful as a biomarker for diagnosis or staging of chronic GVHD. Further evaluation on larger numbers of samples is required to confirm these observations.
Donor Leukocyte Infusion after Stem Cell Transplantation in Patients with Juvenile Myelomonocytic Leukemia.
Authors: Yoshimi Ayami , Bader Peter , Matthes-Martin Susanne , Starý Jan , Klingebiel Thomas , Duffner Ulrich , Locatelli Franco , Révész7 Tom , Niemeyer Charlotte M.
Published: April 2020

Juvenile myelomonocytic leukemia (JMML) is a rare clonal disorder of early childhood. Currently, only allogeneic stem cell transplantation (SCT) offers long-term cure. Relapse remains the major cause of treatment failure. Although graft-versus-leukemia (GVL) effect most likely plays an important role in controlling JMML, the benefit of donor leukocyte infusion (DLI) following SCT in JMML is currently unknown.

Patients and methods: Twenty-one patients with JMML who received DLI after SCT, including 4 patients after given a second SCT, were studied (BMT 14, PBSCT 6, CB 1). The median age at SCT was 15 (8–99) months. A normal karyotype, monosomy 7 or other aberrations were observed in 15, 2, and 4 patients, respectively. Six patients were transplanted from a matched sibling and 15 from an alternative donor. Chimerism analyses were performed by microsatellite PCR system or FISH for sex mismatch in all the patients. Response was defined as the achievement of complete chimerism (CC) and no evidence of hematological relapse. DLI was given either for the development of mixed chimerism (MC) in 7 patients (MC group) or for cytogenetic/hematological relapse in 14 patients (relapse group). Prior to DLI, cyclosporin A had been stopped in all patients, and no child had received chemotherapy.

Results of DLI: Five of the 21 patients received a single DLI, 16 patients 2–6 infusions (median 3). The total T cell dose given ranged from 9x104 to 2.4 x108/kg. Six of 21 patients responded: 3 of 7 patients in the MC group and 3 of 14 patients in the relapse group. The infusion of at least 1x107/kg T cells was needed for durable response. Response was observed in all karyotype subgroups. None of the 6 patients receiving DLI from a matched sibling responded. Five patients developed acute GVHD following DLI and 4 of them responded to DLI. On the contrary, only 2 of the 16 children who did not show acute GVHD after DLI responded. Chronic GVHD developed in 2 responders. The outcome of even the responders was unfavorable. Only one of the responders is alive in remission, with severe chronic GVHD, 72 months after DLI. Two patients relapsed 26 days and 54 months after DLI (one as gastric chloroma), and 3 died of complications of DLI (acute GVHD, bone marrow aplasia, and hyper-eosinophilic syndrome). Four non-responders and one responder with subsequent relapse were rescued by a second SCT.

Conclusion: This study shows that DLI can induce a GVL effect in some of the JMML patients. However, the benefit of DLI in our series of patients was limited because of severe complications in responders and lack of a durable effect. Some modification of DLI, with previous cytoreduction by chemotherapy or a novel drug such as E21R and concomitant administration of cytokines such as interferon alpha, can possibly improve the result of DLI.
Safety and Efficacy of Large Volume Leukapheresis for Allogeneic Hematopoietic Progenitor Cell Transplantation- the University of Miami Experience.
Authors: Shariatmadar Sherry , Nassiri Mehdi , Goodman Mark , Pereira Denise L. , Fernandez Hugo F.
Published: April 2020

Purpose: To report our experience with large volume leukapheresis (LVL) for allogeneic hematopoietic progenitor cell (HPC) transplantation, evaluating its safety and efficacy in 69 consecutive healthy donors.

Methods: Donor medical records from 1995–2004 were reviewed. These consisted of 38 males, 31 females, age range 16–65 (median: 37). All were mobilized with G-CSF, based on a dose of 7.5 mcg/kg/day (median: 600 mcg, range 300–960 mcg) for four days prior to outpatient LVL (Cobe spectra). Vascular access was femoral vein in 59 and antecubital veins in 10 donors. Pre- and post-LVL determinations included: WBC count, hemoglobin, hematocrit, platelet count, serum potassium, and calcium (Ca++). Oral KCL and oral/intravenous Ca++ were given as indicated by laboratory values and symptoms. Catheter-related complications and blood product requirements associated with collection were assessed. Final CD34/kg recipient body weight (rec. BW) and recipient engraftment data was evaluated.

Results: LVL was well tolerated by all donors undergoing a total of 74 procedures.

Data/LVL Procedure Mean Range Procedures per patient 1 1–2 Blood volume processed (L) 24.2 15.0–34.4 Time (minutes) 273 180–386 Flow rate (ml/min) 92 65–100 ACD–A used (ml) 1008 636–1449 Heparin used (units) 3000 Not applicable CD34/kg rec. BW 8.2 1.36–33.9

Adverse reactions were minor and occurred in 40% of the donors. Local parasthesias accounted for majority (86%) of the adverse events. Bone pain secondary to G-CSF accounted for the remainder of the cases and was well controlled with analgesics. Oral electrolyte supplements were administered in 61% of the donors. Eleven (15.9%) required intravenous Ca++ during collection. There were no catheter-related complications and no requirement for blood products during or after collection. Target values of 2 x 106 CD34/kg rec. BW was obtained in 68 cases (98.6%). Engraftment was achieved in 92% of the cases (6 died prior to engraftment). There were no early or late graft failures. Median time to ANC recovery was 10 days (range 8–25 days). Self-sustained platelet count was achieved in a median of 13 days.

Conclusion: LVL was a safe and efficacious method of allogeneic HPC collection. The use of a lower G-CSF dose for mobilization is more economical and well tolerated. This dose allows for adequate collection of CD34 positive cells. These cells support the sustained recovery from high-dose chemotherapy for allogeneic transplantation.