bluuebunny/crossref_metadata_2025 huggingface dataset:
Female Gonadal Function Following Reduced Intensity Conditioning (RIC) Allogeneic Bone Marrow Transplantation for Haematological Malignancies.
Authors: Kyriakou Chara , Davies Melanie , Chatterjee Ratna , Castleton Anna , Roberts Kim , Peniket Andy , Mackinnon Stephen , Kottaridis Panagiotis D.
Published: October 2019

It is generally accepted that recipients of myeloablative allogeneic bone marrow transplants will have markedly reduced gonadal function post transplantation and are likely to be rendered infertile. Many of these patients will also have received intensive pre-transplant chemotherapy leading to significant gonadotoxicity even before transplantation. During the last decade, conditioning regimes of reduced intensity have been developed in an effort to reduce the short and long term toxicities associated with myeloablative transplants,. We studied pre and post-transplant gonadal function of 27 female patients undergoing RIC using Fludarabine, Melphalan and CAMPATH-1H regimen. GVHD prophylaxis consisted of cyclosporine (CyA) except one patient with AML who received CyA/MTX rather than CAMPATH-1H. The median age at transplantation was 33 years (19–45). Fourteen patients had Hodgkin lymphoma, 6 Non Hodgkin lymphoma, 4 multiple myeloma 2 MDS and one AML. Of these patients, 22 were shown to have reduced gonadal function prior to transplantation, as a result of previous intensive chemotherapy (11 patients had previous autologous transplant). The remaining five patients were shown to have preserved gonadal function pre-transplantation as evidenced by LH, FSH, oestradiol and prolactin levels within the normal range. One patient with myeloma had received VAD chemotherapy prior to transplant, one patient with lymphoma CHOP-R × 6, one with AML standard induction anthracycline based chemotherapy while 2 patients with MDS were allografted with no preceding chemotherapy. Post transplantation, all five patients were shown to have preserved gonadal function at a median follow up of 22 months (8–35). This was evidenced by clinical (regular menstruation), biochemical (normal hormonal profile), and radiological (ovarian ultrasonography) parameters. One patient become pregnant naturally twice and gave birth to two healthy children. In contrast to patients receiving fully myeloablative conditioning regimes, female patients with normal gonadal function who receive reduced intensity conditioned allografts can retain gonadal function and fertility. These results have implications in fertility counselling and should be taken into consideration in decision making, especially in young women for whom preservation of fertility is of paramount importance.
Flow Cytometric Analysis of Specimens by a Central Reference Laboratory in a Multi-Center Study: Factors Affecting Data Quality.
Authors: Durett April G. , Gee Adrian P. , Collins Nancy H. , Eapen Mary , Weisdorf Daniel
Published: October 2019

Central reference flow cytometry laboratories are used in multicenter trials to eliminate inter laboratory variation in testing. Yet, factors that materially affect the quality of data such as graft processing, sample composition, packaging, and shipment have been little studied. The Baylor Center for Cell and Gene Therapy (BCGT) was the central reference laboratory for the NMDP IRB-2002-0068 multicenter study on the correlation of graft composition on clinical outcome. Samples of processed or non-processed grafts of 489 bone marrow (BM) and 696 peripheral blood progenitor cell (PBPC) grafts from July 2003-March 2004 were shipped to BCGT using 2 types of shipping containers with frozen gel packs to keep samples cool during transit. On arrival samples were assessed for temperature of gel packs, sample integrity (clumps, clots requiring filtration), viability by flow cytometry using 7AAD, and immunophenotype (CD34+, CD3+, and CD3+ T-cell subsets, CD25, CD69, HLA-DR). The FDA “standard” of 70% viability was used to define acceptable sample viability. Integrity and viability of BM and PBPC samples were not influenced by the method of cell processing: non-processed, plasma depletion, mononuclear cell concentration or T-cell depletion. BM samples were not affected by either temperature or time to evaluation. Viability of PBPC samples was adversely affected by both gel pack temperature and time from collection to evaluation. A 55% mean viability was observed with gel packs arriving thawed-warm vs 89% with frozen gel packs. Additionally, the samples that arrived >48hr after collection also had mean viability less than the 70% standard value. Viability of samples was inversely correlated with gel pack temperature on arrival and time from collection to evaluation. Therefore, the quality of FACS data and by implication, the graft utility was largely influenced by cell type, temperature in transit, and the time from collection to evaluation. Thus, while the use of a central reference laboratory can produce reliable data on cells following a variety of processing methods, certain factors must be optimized in order to collect data that accurately reflects the samples under study. Shipping standards to maintain graft viability and reliability of laboratory assessments of graft composition must be established and validated against the success of transplantation outcomes.
DNA Repair Genes Are Upregulated in Multiple Myeloma (MM) Patients Relapsing after Tandem Transplantation.
Authors: Tricot Guido , Zhan Fenghuang , Huang Yongsheng , Barlogie Bart , Shaughnessy John
Published: October 2019

Background: Repair of DNA interstrand crosslinks (ICLs) is considered the major mechanism by which resistance to high and low dose melphalan emerges (Spanswick et al. Blood2002; 100: 224). Enhanced ICL repair occurs via the Fanconi anemia (FA)/ BRCA pathway in myeloma cell lines (Chen et al. Blood2005; 106: 698). The aim of this study was to evaluate if DNA repair genes were indeed upregulated at relapse when compared to baseline in patients enrolled on our Total Therapy 2 protocol.

Methods: We compared 51 paired baseline and relapse bone marrow samples for gene expression profiling of CD138 selected plasma cells using the Affimetrix U133 Plus 2.0 microarray. The microarrays were preprocessed using GCOS 1.1 software and normalized using conventional GCOS 1.1 scaling. Baseline and relapse gene expression was compared using the paired Student t test.

Results: More than 30 genes related to DNA repair on the microarray were analyzed. We observed significantly higher expression at relapse of FANCA (p=.003), BRCA 1 (p<.001), CHEK1 (p=.006) and FANCG (p=0.03) (Figure 1). In contrast, FANCD2 was down-regulated at relapse (p=.006), while no significant changes in expression of FANCF or DNA mismatch repair genes were seen.

Conclusion: Our data supports the concept that DNA repair genes in the FA/BRCA pathway contribute to acquired resistance to melphalan in patients after high dose melphalan transplants and that inhibition of DNA repair by bortezomib, gemcitabine or the CHEK1 kinase inhibitor UCN-01, may reverse such resistance if added to high dose melphalan.

DNA Repair Genes in Paired Relapse Samples (N=51) DNA Repair Genes in Paired Relapse Samples (N=51)
CKS1B, Mapping to an Amplicon on Chromosome 1q21 and over Expressed in Aggressive Disease, Regulates Multiple Myeloma Growth and Survival through p27Kip1-Dependent and Independent Mechanisms.
Authors: Zhan Fenghuang , Colla Simona , Chen James B. , Stewart James P. , Wu Xiaosong , Huang Yongsheng , Hanamura Ichiro , Burington Bart , Yaccoby Shmuel , Epstein Joshua , Barlogie Bart , Shaughnessy John D.
Published: October 2019

While deregulated expression of a D-type cyclin is thought to represent an initiating event in myelomagenesis, the molecular mechanisms of disease progression are not understood. We recently reported that disease progression and high-risk disease are strongly correlated with increased copy number and expression of genes mapping to chromosome 1q. Here we report that elevated expression of CKS1B, a regulatory subunit of the SCF-Skp2 ubiquitin ligase that regulates p27Kip1 protein stability, mapping to an amplicon at 1q21 and over expressed in high-risk myeloma, is required for myeloma cell survival both in vitro and in-vivo. CKS1B expression, absent in plasma cells from healthy individuals and the benign plasma cell dyscrasia monoclonal gammopathy of undetermined significance, is inversely correlated with p27Kip1 protein levels in primary MM. Silencing of CKS1B in MM cell lines JJN3 and OCI-MY5 using shRNAs delivered by constitutive and inducible lentivirus vectors induced stabilization of p27Kip1, cell cycle arrest and apoptosis. Over expression of a non-degradable form of p27Kip1 in JJN3 and OCI-MY5 cells inhibited cell cycle progression, but did not induce apoptosis. We propose that CKS1B may play in important role in myeloma progression through p27Kip1-related and unrelated mechanisms.
Modulation of the Mitochondrial (Type 2) Apoptotic Pathway Can Enhance Apo2L/TRAIL - Mediated Killing of Human Multiple Myeloma (MM) Cells.
Authors: Khong Tiffany , Yeh Sung Lin , Souris Karly , Spencer Andrew
Published: October 2019

We and others have previously demonstrated that Apo2/TRAIL effectively induces apoptosis of human myeloma cell lines (HMCL) but exhibits only modest activity against primary MM cells derived from patients with advanced MM. A commonly acquired drug-resistance mechanism found in these patients is the over-expression of the anti-apoptotic protein Bcl-xl. Based on these observations and the potential of Apo2/TRAIL as a specific anti-MM therapeutic agent we have investigated further the mechanism(s) of apoptosis induction in MM and whether inhibition of bcl-xl expression enhances the efficacy of Apo2L/TRAIL induced killing of MM cells. Four HMCL selected on the basis of high (U266 and RPMI 8226) or low (LP-1 and NCI H929) Bcl-xl expression, as determined by both Western blotting and ELISA, were chosen for study. We have previously demonstrated no correlation between the absolute levels of caspase 8 and cFLIP or the caspase 8:cFLIP ratio of these 4 HMCL and their sensitivity to Apo2/TRAIL. HMCL were treated with 1μg/ml of recombinant human trimeric TRAIL (LZ-TRAIL) (Amgen) and the level of apoptosis was measured by AnnexinV expression at 2 hours - U266 24%, RPMI 8226 53%, LP-1 57% and NCI H929 26%. The experiment was then repeated and both whole cell lysates and cytosolic fractions were prepared for immunoblot analysis of caspase 8 cleavage and cytochrome C/SMAC protein levels, respectively, at various time-points from 0 to 120 minutes following treatment. Two patterns of apoptosis induction were observed. All 4 HMCL had demonstrable increases in cytosolic cytochrome c and SMAC by 15 minutes post LZ-TRAIL. Similarly both RPMI and LP-1 demonstrated contemporaneous cleavage of caspase 8. In contrast, no evidence of caspase 8 cleavage was seen in U266 or NCI H929 until 30 – 60 minutes post LZ-TRAIL. The impact of pre-treatment with a caspase 9 inhibitor, (Z-LEHD-FMK) prior to LZ-TRAIL correlated with the immunoblot findings: RPMI 8226 and LP-1 (7% and 20%, respectively) showing less relative reduction in apoptosis inhibition than U266 and NCI H929 (64% and 49%, respectively). Finally, treatment with TRAIL alone, the bcl-xl inhibitor 2MAME (10, 15 or 20μg/ml) alone or both (2MAME 4 hours prior to TRAIL) demonstrated synergistic killing of both ’high’ bcl-xl expressing HMCL (RPMI 8226 synergism quotients = 1.1, 1.3 and 1.3 and U266 synergism quotients = 1.6, 1.4 and 1.4 for 2MAME 10, 15 and 20μg/ml, respectively) but not the ’low’ bcl-xl expressing HMCL (NCI H929 and LP-1). From these data we conclude that human MM cells exhibit variable apoptotic responses to Apo2L/TRAIL that involve both the Type 1 and Type 2 apoptotic pathways and that the targeting of known inhibitors of the Type 2 pathway can enhance Apo2L/TRAIL-induced apoptosis.
JS-K, a GST-Activated Nitric Oxide Generator, Induces DNA-Double Strand Breaks and Inhibits Growth and Survival of Multiple Myeloma Cells In Vitro and In Vivo.
Authors: Kiziltepe Tanyel , Hideshima Teru , Raje Noopur , Ishitsuka Kenji , Ocio Enrique M. , Catley Laurence , Li Chun-Qi , Trudel Laura , Yasui Hiroshi , Shirashi Norihiko , Tai Yu-Tzu , Chauhan Dharminder , Mitsiades Constantine , Saavedra Joseph E. , Wogan Gerald N. , Keefer Larry K. , Shami Paul J. , Anderson Kenneth C.
Published: October 2019

JS-K (O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) is a diazeniumdiolate class of prodrug which is designed to release nitric oxide (NO·) on reaction with glutathione S-transferases (GST). GST has been shown to be overexpressed in a broad spectrum of tumor cells. Therefore, JS-K can possibly turn GST overexpression to the tumor’s disadvantage by generating high intracellular concentrations of cytotoxic NO·. Multiple myeloma (MM) is currently an incurable hematological malignancy where new treatment options are urgently needed. In this study we investigated the cytotoxicity of JS-K in MM in vitro and in vivo. JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient MM cells (IC50: 0.3–2.5 mM). Importantly, no significant cytotoxic effects of JS-K at these doses were observed in normal peripheral blood mononuclear cells. JS-K treatment induced apoptosis in MM cells which was associated with PARP, caspase 8, and caspase 9 cleavage; increased cell surface expression of Fas/CD95; Mcl-1 cleavage; Bcl-2 phosphorylation; as well as mitochondrial cyt c, AIF, and EndoG release. Moreover, JS-K could overcome the survival and growth advantages conferred by exogenous IL-6 and IGF-1, or by adherence of MM cells to bone marrow stromal cells. Flow cytometry experiments revealed significant NO· generation in JS-K-treated MM cells. Since NO· is known to cause DNA double strand breaks (DSB), we hypothesized that JS-K induces DSB in MM cells and confirmed DSB formation by neutral comet assay. We further showed that JS-K also activated DNA damage response pathways as evidenced by H2AX, Chk2 and p53 phosphorylation. In addition, JNK was also activated by JS-K treatment in MM cells, and inhibition of JNK significantly decreased JS-K-induced cytotoxicity, suggesting that JS-K induced apoptosis is mediated via JNK signaling. Finally, JS-K was also significantly effective in inhibiting tumor growth and prolonging median survival (p < 0.01) in a human plasmacytoma xenograft mouse model. Analysis of tumors harvested from treated animals showed that JS-K induced apoptosis and decreased angiogenesis in vivo. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.
Mi-63, a Small Molecule Inhibitor of MDM2-p53 Interaction, Has Significant In Vitro Activity in Multiple Myeloma.
Authors: Kumar Shaji , Timm Michael , Kline Michael P. , Haug Jessica L. , Kimlinger Teresa K. , Yang Dajun , Rajkumar S. Vincent
Published: October 2019

Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma.

Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells.

Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.
Molecular Prognosis in Multiple Myeloma: The IFM Experience.
Authors: Decaux Olivier , Magrangeas Florence , Lode Laurence , Charbonnel Catherine , Gouraud Wilfried , Moreau Philippe , Harousseau Jean-Luc , Attal Michel , Bataille Regis , Campion Loic , Loiseau Herve Avet , Minvielle Stephane
Published: October 2019

Multiple myeloma is the second most common hematological malignancy and remains incurable despite high dose chemotherapy. Patients experience survival periods of few months to more than ten years. We used gene-expression profiles of malignant plasma cells obtained at diagnosis to develop and validate biomarkers to predict disease prognosis. Gene-expression profiling was realized on 250 bone marrow samples obtained from patients less than 66 years, treated according to the Intergroupe Francophone du Myelome (IFM) 99 clinical trials, based on double intensive treatments. The median follow-up time was 3 years and 55 patients died from their disease during this period. Supervised methods were performed to identify expression signatures associated with survival in a training group of 182 patients. From these genes, a classifier of prognosis was developed in the training group and validated in an independent validation group of 68 patients. Univariate Cox proportional hazards model at a significant level of p<0.05 and selection procedure from BRB ArrayTools at a significant level of p<0.001 were used to select 50 genes associated with survival. Resampling and permutation tests were performed in an rigorous internal validation procedure and 28 genes with a permutation p<0.005 and a resampling mean p<0.01 were retained. To reduce gene list instability, multiple training/test partitions (100) were performed and the 15 most stable genes were used to build a survival predictor. For each patient, a score based on the first component of principal component analysis was calculated. The patients were ranked according to their score and divided according to the median (low-risk and high-risk). The model was highly significantly associated with survival in the training group (p<0.001) and it was confirmed in the validation group (p<0.001). The relative risk of death for high-risk patients was 8.46 (95% confidence interval (CI) = 3.3 to 21.6) in the training group and was 15.3 (95% CI = 2 to 116.5) in the validation group. Various biological variables including serum beta2-microglobulin, serum albumin, combined in the International Staging System (ISS), the deletions/monosomy of chromosome 13 (del13) and the t(4;14)(p16;q32) have emerged as relevant prognostic factors in MM. The 15-gene prognostic classifier was independent of ISS (p<0.001) with an adjusted hazard ratio of death =7.5 (95% CI = 3.09 to 18.2), of del13 (p<0.001) with an adjusted hazard ratio of death =8.8 (95% CI = 3.7 to 21), and of t(4;14) (p<0.001) with an adjusted hazard ratio of death =9.3 (95% CI = 3.9 to 22.1). These results demonstrate that this molecular predictor is very powerful to identify different categories of patients based on expected survival, and thus may be clinically useful to design therapeutic trials.

Figure Figure
CCR1 Inhibition Impairs Osteoclast Activity and Interaction with Myeloma Cells.
Authors: Vallet Sonia , Raje Noopur , Ishitsuka Kenji , Hideshima Teru , Podar Klaus , Kizeltepe Tanyel , Yasui Hiroshi , Chhetri Shweta , Ocio Enrique M. , Jin Janice , Okawa Yutaka , Ikeda Hiroshi , Boccadoro Mario , Anderson Kenneth C.
Published: October 2019

Multiple Myeloma (MM) is characterized by increased osteoclasts (OC) activity leading to severe bone disease. Several inducers of OC number and activity have been identified, including several key chemokines. In particular, macrophage inflammatory protein-1α (MIP-1α) and RANTES activate CCR1 and CCR5, resulting in increased osteoclastogenesis (Oba et al. Exp Hematol. 2005) and increased OC motility (Yu X. J Bone Miner Res. 2004) These studies underscore the role of CCR1 in promoting osteoclastogenesis. Here we demonstrate the effects of inhibition of CCR1 with a specific receptor antagonist (MLN3897, Millenium Pharmaceuticals) on normal mature OC. Mature OC were obtained by stimulating PBMC of healthy donors with RANKL and MCSF (50 ng/ml) for three weeks. OC expressed high levels of CCR1 and secreted both MIP-1α (1 ng/ml +/− 1.8) and RANTES (12 pg/ml +/− 0.66), suggesting an autocrine effect of these chemokines. Analyzing the bone resorptive ability with a pit formation assay, we observed that MLN3897 impaired OC function in a dose-dependent fashion (at 10 nM: 20% of reduction in resorbed area, at 100 nM: 50% of reduction). We then studied the effects of CCR1 inhibition on OC viability by analyzing nuclear integrity with Hoechst33258 staining. After 12 hours of cytokine-deprivation, MLN3897 enhanced OC nuclear fragmentation and condensation, suggesting a role for CCR1 ligands in OC survival. We next studied the interactions between OC and MM cells. Because OC secrete several chemoattractants, we studied their ability to stimulate RPMI8226 MM cell migration. We first confirmed high expression levels of CCR1 on RPMI8226 MM cells by flow cytometry (86% compared to isotype control). Inhibition of CCR1 with MLN3897 did not induce any direct cytotoxicity or growth arrest of these cells. We then studied the effects of OC on RPMI8226 MM cells. We observed that OC potently stimulated migration of RPMI8226 MM cells (8-fold increase), which was almost completely blocked by treatment with MLN3897. In contrast, MIP-1α alone induced only a modest effect on migration: the highest effective concentration (0.5 ng/ml) induced only a 2-fold increase. Neutralizing MIP-1α antibody partially reversed these effects, suggesting that other factors may contribute to this migratory effect. Our data therefore demonstrate that CCR1 inhibition by MLN3897 impairs mature OC survival and activity. Although MLN3897 does not have any direct antiMM activity, it affects MM-OC interactions and inhibits MM cell migration to OC. Ongoing studies are further characterizing the effects of CCR1 inhibition on MM-OC interactions in order to provide the framework for its clinical evaluation in MM.
Differentiation of Multiple Myeloma and MGUS by Cross Validated Differential Proteomic Analysis.
Authors: Brown Ross D. , Belov Larissa , dos Remedios Cris , Sze Daniel , Cooper Simon , Dolotin Mila , Gibson John , Joshua Douglas E.
Published: October 2019

The recent development of a standardised proteomic microarray technique, DotScan, has allowed an innovative approach to the investigation of haematological disorders. In this study, mononuclear cells from 49 peripheral blood samples were studied to determine whether the technology could identify a differential consensus pattern of antigen expression for patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). An automated reader simultaneously determined the expression of 82 different cell surface antigens by binding cells to antibodies on microscopic dots on the nitrocellulose-coated region of a microscope slide. The figure illustrates the image from a typical slide. A consensus pattern of antigen expression was analysed in duplicate by cross-validated discriminant analysis and a pilot database of disease groups was accumulated. Cross validated discriminant analysis successfully predicted patients with monoclonal gammopathy (98% success) and a discrete mosaic pattern could differentiate patients with MM who were treated with thalidomide (n=9). As expected, no single antigen could be used to discriminate between multiple myeloma (n=24), MGUS (n=14) and normal controls (n=11). Antigens with the highest ranking for differentiating the monoclonal gammopathies were CD25 (reduced after thalidomide), CD8 and CD57 (high in MM), CD28 (reduced in MM) and CD95 (reduced in MGUS) reinforcing the importance of immunomodulatory mechanisms in both MM and MGUS. Traditional flow cytometry was used to confirm these specific observations but also to demonstrate that the reduced CD28 expression was specific for CD8+ cells and the reduced CD95 expression was on CD8+ CD57+ cells. Three patients with MGUS were misclassified as MM but on review these 3 patients could have been classified as smoldering myeloma. There was no significant difference in the mosaic of 5 long term survivors of MM (> 10 years). This study has demonstrated the potential of using disease-specific databases to compare the mosaic of antigen expression for the diagnosis of monoclonal gammopathies. Long term studies will be required to accurately determine the prognostic significance at diagnosis and the ability of consensus patterns to identify which MGUS patients develop MM.

Figure Figure Specificity and sensitivity of proteomic array for monoclonal gammopathies MGUS MM MM Thal Normal Sensitivity % Specificity % MGUS prediction 5 3 2 4 35 100 MM prediction 0 15 0 0 100 91 MM Thal prediction 0 0 9 0 100 95 Normal prediction 0 0 0 11 100 89
Distinct Roles of Integrins α6 and α4 in Fetal Liver Hematopoietic Stem and Progenitor Cell Homing.
Authors: Qian Hong , Georges-Labouesse Elisabeth , Nyström Alexander , Domogatskaya Anna , Tryggvason Karl , Jacobsen Sten Eirik W. , Ekblom Marja
Published: October 2019

During development and after transplantation, intravenously injected hematopoietic stem and progenitor cells (HSPCs) selectively transmigrate through the sinusoidal walls into the bone marrow (BM) niches to engraft and reconstitute hematopoiesis. The rate of reconstitution following transplantation varies depending on the source of hematopoietic stem cells (HSCs) (To, et al 1992). However, the molecular pathways that control the homing of HSCs, in particular, of fetal HSCs are still not well understood. In the present study we studied the contribution of α6 and α4 integrins in homing of fetal liver HSPCs into adult BM by using function-blocking antibodies and an integrin α6 knockout mouse model. We found an ubiquitous expression of both integrin α6 and α4 receptors on fetal liver Lin−Sca-1+c-kit+ (LSK) HSPCs. Genetic ablation of integrin α6 resulted in reduced homing of fetal liver progenitors (HPCs) to BM of lethally irradiated adult recipients. In agreement with this, the integrin α6 antibody inhibited homing of fetal liver HPCs into BM and spleen. The role of integrin a6 in homing and engraftment of fetal liver HSCs was studied by a competitive repopulation assay by using integrin α6−/− or α6+/+ fetal liver cells. Absence of α6 integrin in fetal liver cells did not cause any engraftment defect or mobilization hypersensitivity as compared to wild-type cells. In agreement with this, anti-integrin α6 antibody did not either inhibit BM homing of short-term or long-term HSCs. In contrast, homing of fetal liver HSCs and HPCs to BM was virtually abrogated after treatment with integrin α4 antibody. Our results show that the α6 integrin receptors are functional during homing of fetal liver HPCs, but not multilineage repopulating HSC in vivo. Furthermore, we show the critical role of integrin α4 receptor for homing of both fetal liver HPCs and multilineage repopulating HSCs to BM, indicating distinct developmentally regulated functions for integrin α6 and α4 receptors during fetal hematopoiesis
Survival Outcomes of Patients Receiving Thalidomide-Dexamethasone for Previously Untreated Multiple Myeloma.
Authors: Thomas Sheeba K. , Giralt Sergio A. , Wang Michael , Delasalle Kay B. , Gavino Maria , Rankin Kim , Alexanian Raymond , Weber Donna M.
Published: October 2019

Background: In three recent Phase 2 and 3 studies, the combination of thalidomide-dexamethasone (TD) induced responses in 64–72% of patients, including complete responses in 4–16% of patients. However, long term outcomes of patients receiving induction therapy with TD have not been well described.

Patients and Methods: In a retrospective fashion, we updated our experience (median f/u of 50 mos.) of 58 patients treated between 7/2000 and 5/2002, with thalidomide at 100mg nightly, increasing by 100mg every 7 days as tolerated to a maximum dose of 400mg (median 200mg), and dexamethasone (20mg/m2) on days 1–4, 9–12, and 17–20 (decreased to days 1–4 only, beginning with cycle 3). Cycles were repeated on day 28 and patients were treated on protocol for a maximum of 4 courses. To prevent thrombosis, 34 patients received therapeutic doses of either warfarin (INR 2–3) or low molecular weight heparin.

Results: Of 58 patients, 31 proceeded to myeloablative therapy followed by autologous stem cell support. Another 16 patients received maintenance chemotherapy until disease progression. The median age at enrollment was 60.5 (37–78), and 60% were male. By International Staging System criteria, 41% of patients were stage 1, 33% stage 2 and 26% stage 3. The overall response rate was 72%, including 12% with CR. Median overall survival (OS) of those who received autologous stem cell transplant (ASCT) exceeds 59.6 months. Median OS of those who did not receive ASCT was 31.8 months. The observed difference in OS may be partially explained by differences in performance status between the 2 groups.

Conclusion: Our survival data with TD induction does not differ significantly from either historical data with alkylating agent based therapy, or from previously noted prolongation of OS after intensive therapy with stem cell support (IFM-90). Further study of the impact of thalidomide and other novel agents as induction therapy in both the ASCT and non-ASCT settings is warranted.
Transvection Mediated by the Translocated Cyclin D1 Locus in B Cell Malignancies.
Authors: Epner Elliot , Huang Jing , Liu Hui , Wang Jin , Fleming W.H.
Published: October 2019

Translocations involving the immunoglobulin heavy chain gene locus (IgH) are common in B cell malignancies. One common target gene is cyclin D1, which is deregulated in most patients with mantle cell lymphoma (MCL) and 20–30% of patients with multiple myeloma (MM). The cyclin D1 locus represents a model locus to study the role of epigenetics in B cell malignancy. Cyclin D1 is known not to be expressed in normal lymphocytes, where the locus is DNA hypomethylated. In genetic variants that have lost the translocated chromosome, the cyclin D1 locus assumes a densely methylated configuration. Reintroduction of the translocated chromosome by somatic cell fusion reverts the methylated normal locus to a hypomethylated state in trans. Thus, the translocated chromosome exerts a trans DNA hypomethylating effect on the normal chromosome that resembles transvection in Drosophila. This is the first evidence of transvection in a human malignancy. Similar to transvection in Drosophila and paramutation in plants, evidence for pairing of the cyclin D1 loci and the participation of small RNA’s in this process has been obtained. Combined FISH/ immunofluorescent studies have shown the colocalization of both the normal and translocated chromosome 11’s at the nucleolus. Using chromatin immunoprecipitation assays, we show that CTCF and nucleophosmin are present at both the normal and translocated chromosomes in B cells expressing cyclin D1 but not in B lymphocytes. Tethering of the translocated and nontranslocated cyclin D1 loci by CTCF/nucleophosmin at the nucleolus provides physical and spatial juxtaposition of the cyclin D1 loci permissive for transvection.
Erythropoietin Therapy and Venous Thromboembolic Events in Patients with Multiple Myeloma Receiving Chemotherapy with or without Thalidomide.
Authors: Zangari Maurizio , Cavallo Federica , Prasad Keshava , Fink Louis , Coon Sharon , Barlogie Bart , Tricot Guido
Published: October 2019

Background: The association between the use of recombinant human erythropoietin (rHUEPO) and venous thromboembolic events (VTE) has been described in cancer patients treated with chemotherapy. A higher risk of VTE has also been observed with the concurrent use of erythropoiesis-stimulating agents and chemotherapy.

Methods: We retrospectively analyzed VTE incidence and concurrent use of erythropoiesis-stimulating agents in patients with multiple myeloma (MM) enrolled in our Total Therapy 2 (TT2) study. TT2 included 4 monthly cycles of induction chemotherapy followed by tandem transplant. Patients were randomly assigned to receive Thalidomide or not during the whole treatment. Both arms otherwise received identical chemotherapy.

Results: The charts of 599 of a total of 668 patients enrolled (90%) were reviewed; 284 patients received Thalidomide, 315 did not. With median age was 57 years, 59% were male, 24% had IgA myeloma. 62% of patients received erythropoietin (erythropoietin alpha 40000 U SQ/week) during the first 4 months of treatment. In thalidomide treated patients, VTE occurred in 33 out of 166 (20%) patients who concomitantly received administration of erythropoietic agents and in 38 out of 118 (32%) patients who did not receive erythropoietin (p=.025). Among patients not receiving thalidomide, VTE occurred in 18 out of 203 (8%) patients who received concomitant administration of erythropoietic agents and in 17 out of 112 (15%) patients who did not receive erythropoietin (p=.10).

Conclusions: The development of thrombosis in patients enrolled on TTII protocol was not increased by the concomitant use of erythropoiesis-stimulating agents and thalidomide. Similarly, erythropoiesis-stimulating therapy did not affect VTE incidence in newly diagnosed myeloma patients treated with intensive chemotherapy without thalidomide.
Plasmapheresis in the Treatment of Renal Failure Associated with Multiple Myeloma.
Authors: Kumar Ambuj , Djulbegovic Benjamin , Soares Heloisa P.
Published: October 2019

Background: Renal failure is a common and severe complication of multiple myeloma. About 20% of the population with myeloma present with renal insufficiency and is associated with increased risk of early mortality. Plasmapheresis has been proposed as a method to reduce rate of permanent renal failure by removing the light chains from the plasma in myeloma patients. However, evidence related to the addition of plasmapheresis to chemotherapy for renal recovery in myeloma patients in existing literature is conflicting. The objective of this study is to present the totality of evidence by conducting a systematic review to assess the efficacy of plasmapheresis in preventing irreversible renal failure in patients with multiple myeloma.

Methods: A systematic and comprehensive search of the literature was performed using MEDLINE, and Cochrane library databases from 1966–2006 for all phase III randomized controlled trials comparing the use of chemotherapy with forced diuresis plus plasmapheresis versus chemotherapy plus forced diuresis in myeloma patients. Additionally, we searched ASCO and ASH meeting abstracts for the last two years. Data were extracted and pooled on overall survival and renal outcome (discontinuation of dialysis, improvement in renal function/creatinine clearence) as per the methods recommended by the Cochrane Collaboration.

Results: Altogether we identified 25 trials out of which 3 studies met the inclusion criteria. Overall, the methodological quality of the trials was good and there was no statistically significant heterogeneity between trials [outcome: overall survival (p=0.2), renal outcome (p=0.6)]. Overall survival and renal outcome were similar for the plasma exchange group and the control. Hazard ratio for the outcome overall survival was 0.76 (95% CI 0.47, 1.25, p=0.3) and for the requirement of dialysis was 0.71 (95%CI 0.35, 1.42, p=0.3) indicating no benefit for plasmapheresis in the treatment of renal failure associated with myeloma (see figure 1).

Conclusion: In multiple myeloma patients with acute renal failure there is no conclusive evidence that plasmapheresis has any effect on overall survival or on the requirement of dialysis.

Plasma exchange in renal failure for myeloma patients Plasma exchange in renal failure for myeloma patients
Role of SH2 Domain in JAK2-V617F Mediated Transformation.
Authors: Gorantla Sivahari P. , Dechow Tobias , Peschel Christian , Duyster Justus
Published: October 2019

A point mutation in JAK2 (V617F) has been described recently in patients with myeloproliferative diseases like polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (IMF). This V617F point mutation in JAK2 has been shown to activate several downstream pathways including STAT5 and ERK. This mutation also renders haematopoietic progenitors cytokine-independent. The role of the V617F mutation in oncogenesis is not fully understood. In this study we aim to dissect the role of the SH2 domain in JAK2-V617F mediated transformation.

Stable Ba/F3 cell lines expressing JAK2-wild type (wt), JAK2-V617F, JAK2-R439K (SH2 domain mutation) and JAK2-V617F/R439K mutants were generated. Cell proliferation assays showed that JAK2-V617F transforms Ba/F3 cells and renders them IL3 independent, while wild type JAK2 and JAK2-R439K could not. Surprisingly, JAK2-V617F/R439K was not able to induce a transformed phenotype in Ba/F3 cells. Imunoblotting revealed strong activation of JAK2, STAT5 and ERK in cells expressing JAK2-V617F, whereas no such activation could be found in JAK2-wt, JAK2-R439K and in JAK2-V617F/R439K expressing cells. Thus the SH2 domain in JAK2-V617F seems to play a crucial role in the transformation of Ba/F3 cells containing a heterodimeric (IL-3) cytokine receptor.

It has been demonstrated that JAK2-V617F induces cellular transformation more efficiently in cells expressing a homodimeric cytokine receptor such as the erythropoetin receptor. We therefore established Ba/F3 cells overexpressing EpoR together with JAK2-wt, JAK2-V617F, JAK2-R439K and JAK2-V617F/R439K. In contrast to parental Ba/F3 cells, EpoR expressing Ba/F3 cells could be transformed by both JAK2-V617F as well as JAK2-V617F/R439K. Both the single and double mutant Ba/F3 cells showed strong activation of STAT5 and ERK. This suggests that an intact SH2 domain is not required for homodimeric cytokine receptor expressing cells.

These results show that transformation by JAK2-V617F requires an intact SH2 domain only in cells expressing a heterodimeric cytokine receptor. In contrast, cells containing a homodimeric cytokine receptor are able to induce transformation in the presence of JAK2-V617F with an additional SH2 mutation. Further progress in understanding the role of the SH2 domain in JAK2-V617F mediated transformation may help in delineating downstream signalling with therapeutic implications.
Activating Shp2 Mutations Cause Progressive Myeloproliferative Disorder in Mice.
Authors: Mohi M. Golam , Chan Gordon , Araki Toshiyuki , Yang Wentien , Pao Lily , Kalaitzidis Demetrios , Neel Benjamin G.
Published: October 2019

Mutations in the SH2-containing tyrosine phosphatase, Shp2 (PTPN11) have been found in human diseases. Germ-line Shp2 mutations cause ~50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ~35% of sporadic JMML, most other cases have either activating Ras mutations or homozygous Nf1 deficiency. Shp2 mutations are also found at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and B-cell acute lymphoblastic leukemia (B-ALL). NS and leukemia-associated Shp2 mutations often affect the same residue, but result in different substitution. We previously showed that leukemia-associated mutants E76K or D61Y, but not wild type (WT) Shp2 could transform bone marrow (BM) or fetal liver cells and give rise to cytokine-independent myeloid colony formation. Transplantation of E76K- or D61Y- (but not parental virus or WT Shp2) transduced BM evoked invasive MPD in most recipients and majority of them died at 6–7 months. We have generated knock-in mice for the Shp2 mutants D61G and D61Y to better understand the effects of these mutations under endogenous promoter control. Whereas D61G is associated with both NS and leukemia, D61Y is only found in leukemia and catalytically more active than D61G in basal state. D61G/+ mice exhibited all major features of NS and a mild, well-tolerated MPD for ~1 year and then progressed to fatal MPD. Constitutive expression of the D61Y heterozygous allele was embryonic lethal. However, when an inducible allele of D61Y (inD61Y) was crossed to Mx-Cre mice and expressed upon pI-pC induction, these mice rapidly developed MPD and ~50% mice died within a period of 4–7 months. Immunophenotypic analysis of bone marrow and spleen of these mice showed marked expansion of Gr-1/Mac-1 positive population indicating that these mice died of infiltrating MPD. Bone marrow macrophages from D61G/+ and inD61Y mice showed increased GM-CSF-evoked proliferation and increased overall tyrosine phosphorylation. Likewise GM-CSF-evoked activation of Erk, Akt, Jak2 and Stat5 were enhanced, as was Ras loading suggesting that leukemogenic Shp2 causes MPD by enhancing both Ras/Erk and Jak2/Stat5 pathways. The results of our retroviral and knock-in models suggest that leukemia-associated Shp2 mutant is sufficient to initiate myeloid leukemogenesis in mice.
Tlr4 Is Monoallelically Expressed in Human BFU-E and CFU-E and May Have Application as a Clonality Marker in Hematological Disorders Due to Somatic Mutations in Both Males and Females.
Authors: Chen George L. , Liu Enli , Swierczek Sabina , Prchal Josef T.
Published: October 2019

Tlr4 is monoallelically expressed in a tissue specific manner in mice (Peireira JP, Girard R, Chaby R, et al. Nat Immunol 4(5): 464–70, 2003). We characterized Tlr4 allelic expression in human BFU-E, CFU-E, and reticulocytes. The alleles were distinguished by a single nucleotide polymorphism (SNP) that resulted in creation of a new Bcc1 restriction enzyme cutting site. Individuals were genotyped for the SNP and only individuals heterozygous for the SNP were used in experiments. Polycythemia vera patients were diagnosed based on clinical presentation, the presence of the JAK2 V617F mutation, and clonality by X chromosome inactivation methods when available. Individual human BFU-E and CFU-E were grown from whole blood mononuclear cell fractions on methylcellulose media. Allelic expression of Tlr4 was assayed after reverse transcription of mRNA, nested PCR amplification of cDNA, and Bcc1 digestion of PCR product.

Normal human BFU-E and CFU-E colonies demonstrated monoallelic expression of both alleles as well as biallelic expression (n=1). Polycythemia vera patient BFU-E and CFU-E colonies demonstrated a similar pattern (n=2). Tlr4 allelic expression in reticulocyte RNA fractions was assayed as outlined above and by quantitative reverse transcription PCR methods. Normal human reticulocyte RNA fractions isolated from whole blood demonstrated biallelic expression (n=4). 3 of 5 polycythemia vera patient reticulocyte RNA fractions isolated from whole blood demonstrated skewed biallelic expression, and 2 of 5 polycythemia vera patients demonstrated monoallelic expression of either allele.

Tlr4 is located on chromosome 9 in humans. The finding of monoallelic expression in the reticulocyte RNA of 2 of 5 PV patients suggests thatTlr4 may be used as a phenotypic clonality marker in males and females, anda subset of polycythemia vera patients can be identified within the diagnostic population defined by clinical history and the JAK2 V617F mutation.If the assumption that clonal proliferation follows somatic mutation is true, then at least 2 mutations are implied in the pathogenesis of polycythemia vera: one by the original demonstration of clonality by X-chromosome inactivation methods, and the other by the demonstration of acquired monoallelic Tlr4 expression in the BFU-E/CFU-E to reticulocyte transition. These data are now followed by a prospective study of Tlr4 expression in a large number of female and male patients to be correlated with quantitative JAK2 V617F measurements.
SCF and IL-5 Synergize with FIP1L1/PDGFRα To Induce Mastocytosis in a Chronic Eosinophilic Leukemia Murine Model.
Authors: Yamada Yoshiyuki , Rothenberg Marc E. , Lee Andrew W. , Al-Moamen Nabeel J.H. , Williams David A. , Cancelas Jose A.
Published: October 2019

FIP1L1/PDGFRα (F/P) fusion protein has been identified as a cause of chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES). Our group has previously described a murine CEL/HES model based on the expression of both F/P and T-cell-dependent overexpression of IL-5 (Yamada Y et al., Blood 2006), which develops a full hypereosinophilic syndrome with eosinophil infiltration into multiple organs similar to that observed in patients with CEL/HES (CEL-like mouse). Patients with F/P+ HES/CEL also display mastocytosis. Since mast cell (MC) development largely depends on c-kit signaling induced by its ligand SCF, we aimed to determine if SCF collaborates with the F/P fusion protein and IL-5 to enhance MC development. The CEL-like mice showed higher levels of MC infiltration in small intestine compared to transplanted mice with IL-5 transgenic HSC/P (control, Table 1). Interestingly, the intestinal MC infiltration of CEL-like mice was primarily associated with MC residing in the lamina propria and intraepithelial locations associated with villi; whereas MC in control mice were primarily in the crypt areas (47-fold higher mast cell levels in villi compared to those of control mice). In addition to the small intestine, skin MC infiltration was also significantly increased in CEL-like mice (Table 1). Notably, F/P+ BM hematopoietic stem cell/progenitor (HSC/P) showed proliferation and MC differentiation in vitro in the absence of cytokines (2.3-fold cell expansion, 54% c-kit+/FcεRIα+ cells) while empty vector (EGFP alone)-transduced (control) HSC/P did not survive in this culture condition. Such an expansion became even higher (36-fold expansion, 90% c-kit+/FcεRIα+ cells and 2,000-fold higher than control HSC/P) in the presence of low-concentration of SCF (10 ng/ml) for 3 weeks in culture. In contrast, culture with no cytokines or low-dose of SCF did not induce any MC development from control HSC/P. Unlike low-concentration of SCF, IL-3 (100 ng/ml), which induces a rapid and significant MC expansion in control cells, impaired MC development of F/P+ HSC/P (129-fold lower expansion at 3 weeks of culture), suggesting that MC proliferation induced by F/P expression may trigger different signaling pathways than during normal MC differentiation induced by IL-3. In summary, the F/P fusion protein induces murine mastocytosis via SCF/c-kit signaling, which is synergistically enhanced by IL-5 overexpression.

Table 1. Tissue Mast Cell Infiltration (Mast Cells/mm2) Control (IL-5 overexpressing) mice CEL-like mice *p<0.05 compared to control; n=4, per group Small Intestine 144 ± 70.2 964 ± 316.5* Skin 129 ± 33.2 208 ± 42.8*