bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence and Risk Factors of Complications in Patients with Thalassemia Intermedia
Authors: Tantiworawit Adisak , Winichakoon Poramed , Hantrakool Sasinee , Chai-adisaksopha Chatree , Rattarittamrong Ekarat , Norasetthada Lalita
Published: October 2019

BACKGROUND

Thalassemia intermedia (TI) or Non-transfusion dependent thalassemia (NTDT) is a mild form of thalassemia and does not require regular transfusion. Many complications can be found in this disease and different from thalassemia major(TM). Most studies mainly focus on beta thalassemia and lack of study in alpha group. This study aims to elucidate the prevalence of complications and identify predictive factors affecting complication of both alpha and beta NTDT patients.

METHODS

We retrospectively reviewed NTDT patients who attended our clinic from 1 January 2012 to 31 December 2013. Medical record was reviewed for complication and clinical data.

RESULTS

One hundred NTDT patients were included in this study, female (60%), median age 38 years (19-78). Alpha thalassemia was the majority population (54%). Interestingly, overall complications were found in 83%. The three most common were extramedullary hematopoiesis (47%), cholelithiasis (35%) and abnormal liver function (29%). The others were endocrinopathy (29%), osteoporosis (17%), pulmonary hypertension (14%), cardiomyopathy (11%), thrombosis (4%), and leg ulcer (2%), respectively. The mean ferritin level was 1,563.46 ng/ml. Forty four and seventy six percent of patients had ferritin level more than 2,500 and 800 ng/ ml, respectively.

The significant risk factors affecting complications in extramedullary hematopoiesis were female with odd ratio 2.76 (95% CI 1.046-7.294, p= 0.040) and hemoglobin level below 8 g/dL with odd ratio 3.08 (95% CI 1.113-8.521, p= 0.03) and the significant risk factors affecting complications in osteoporosis were female with odd ratio 7.64 (95% CI 1.514-38.604, p= 0.014) and age more than 40 years with odd ratio 4.66 (95% CI 1.313-16.506, p= 0.017). Iron overload (ferritin > 800 ng/ml) was the only risk factor for abnormal liver function from this study with odd ratio 3.79 (95% CI 1.033-13.919, p= 0.035), but trend to be statistical significant in other complications. Three patients were death and all of them died from sepsis.

CONCLUSION

Alpha thalassemia is the common type of NTDT in our study which is different from previous study. The complication in NTDT is also different from TM. The most common complications were extramedullary hematopoiesis, cholelithiasis and abnormal liver function. The prevalence of iron overload was high in these patients. Thus, regular iron monitoring and early investigation for complication detecting were necessary in this group of patients.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Processing of Neutrophil α-Defensins Does Not Rely on Serine Proteases in Vivo
Authors: Glenthoj Andreas , Nickles Katrin , Cowland Jack B , Borregaard Niels
Published: October 2019

The α-defensins human neutrophil peptides (HNPs) are the predominant antimicrobial peptides of neutrophil granules. They are synthesized in promyelocytes and myelocytes as proHNPs, but only processed and stored as mature HNPs in promyelocytes. Despite decades of search, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unidentified. Thus, neither the enzyme processing proHNPs nor the localization of processing has been identified. It has long been hypothesized that proHNPs are processed by the serine proteases highly expressed in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which have shown in vitrocapability to process recombinant proHNP into HNP.

We investigated, whether serine proteases are in fact responsible for proHNP processing in human bone marrow cells and in human and murine myeloid cell lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of 35S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease proHNP processing by primary human neutrophil precursors. Furthermore, introduction of NE, CG, and PR3 into murine promyelocytic cells did not increase processing capability. Finally, two patients suffering from Papillon–Lefèvre syndrome, who lack the ability to activate neutrophil serine proteases, demonstrated normal levels of fully processed HNP in peripheral neutrophils.

Contradicting earlier assumptions, our study found serine proteases dispensable for processing of proHNPs in vivo. This calls for study of other protease classes in the search for the proHNP processing protease(s).

Disclosures

No relevant conflicts of interest to declare.
12-HETrE, a Novel 12-LOX Oxylipin, Prevents Platelet Activation in a Gαs-like Manner
Authors: Yeung Jennifer , Fernandez-Perez Pilar , Vesci Joanne , Holman Theodore R , Ren Jin , Holinstat Michael
Published: October 2019

Platelet-mediated thrombosis is the primary underlying mechanism leading to cardiovascular life-threatening clinical events. Control of excessive platelet responses is an essential aspect of antithrombotic therapy. A number of anti-platelet drugs have been developed to target specific signaling pathways or endpoints involved in platelet activation. Despite the effectiveness of current anti-platelet therapies, uncontrolled thrombosis or bleeding complications still persist. We had proposed a potential novel therapeutic approach by which oxylipins generated by 12-lipoxygenase (12-LOX) oxidation of ω-6 could modulate platelet reactivity. We observed 12-hydroxyeicosatrienoic acid (12-HETrE), a 12-LOX oxidized oxylipin of ω-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), significantly attenuated human platelet activation. We then verified that DGLA oxidation to 12-HETrE depended on functional platelet 12-lipoxygenase (12-LOX) in our transgenic mouse model deficient in 12-LOX enzyme in the platelets (12-LOX-/-). To determine whether 12-HETrE could be inducing its inhibitory regulation in a GPCR-like manner by which it could potentially be behaving similarly to prostacyclin to activate adenylyl cyclase and increase cAMP through the Gs pathway, we measured cAMP level in the presence of 12-HETrE. We observed 12-HETrE significantly increased cAMP levels. We investigated a downstream effector of cAMP, such as VASP 157 phopshorylation, which is a PKA substrate. Also observed both Rap1 and GPIIbIIa activation to be attenuated in the presence 12-HETrE, confirming our aggregation result. This the first study to show the signaling mechanism of 12-HETrE which is dependent on active 12-LOX oxidation of DGLA to regulate platelet reactivity in a Gs-like manner.

Disclosures

No relevant conflicts of interest to declare.
Anti-Factor VIII C1 Domain Antibodies Are Present in the Plasmas of Patients with Hemophilia and Inhibitors
Authors: Batsuli Glaivy , Cox Courtney , Healey John F. , Lollar Pete , Meeks Shannon L
Published: October 2019

Hemophilia A is an X-linked disorder characterized by a deficiency or absence of blood coagulation protein factor VIII (fVIII). Treatment involves replacement of fVIII through infusions for acute bleeding episodes or prevention of bleeding events. Approximately 30% of individuals with severe hemophilia A will develop antibodies to fVIII. Many studies have characterized the antigenic properties of the C2 and A2 domains as these domains are considered the predominant immunogenic domains of the fVIII protein. However, there is increasing evidence that the C1 domain contributes to fVIII function and immune response to fVIII.

Our laboratory has produced and purified a murine IgG2ak anti-human C1 domain monoclonal antibody (MAb), designated 2A9. In this study, we characterized the functional properties of MAb 2A9 using standard coagulation testing including its anti-fVIII inhibitor titer by Bethesda assay and its ability to inhibit fVIII binding to von Willebrand factor (VWF) by competition ELISA. In the Bethesda assay, 2A9 has an inhibitor titer of 97 BU/mg and is a type II inhibitor. In addition, MAb 2A9 inhibits fVIII binding to VWF in an ELISA assay with a 50% inhibitory concentration (IC50) of 1 µg/ml. This is in comparison to the potent high-titer inhibitory anti-C2 MAb I89 (IC50 0.02 µg/ml) and a control non-inhibitory anti-A2 MAb ID4 (IC50 > 10 µg/ml).

We tested 11 plasma samples from patients with congenital hemophilia with inhibitors in the Emory IRB-approved inhibitor bank. The plasma samples have inhibitory titers ranging from 1 - 188 BU/ml with a median inhibitory titer of 54 BU/ml and mean inhibitory titer 59 BU/ml. The plasmas were tested for the presence of antibodies that compete with anti-C1 domain MAb 2A9 using competition ELISA with fVIII as the antigen. Biotinylated MAb 2A9 was serially diluted and the concentration of antibody required to produce an absorbance at 405 nm of 0.3 was compared between control severe hemophilia A plasma and inhibitor plasma samples. Inhibitor plasma samples that reduced the ELISA titer of MAb 2A9 were considered a competitive inhibitor. Of the 11 inhibitor plasma samples, 4 were found to compete with MAb 2A9.

Our study demonstrates that anti-C1 domain antibodies are present in the plasma of patients with hemophilia A and inhibitors. Given the increasing evidence that the C1 domain is important in fVIII function it is likely that these anti-C1 antibodies are clinically relevant. Therefore, domains other than A2 and C2 need to be included in future studies of fVIII B cell epitopes.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Prevalence of Venous and Arterial Thrombosis Among 268 Heterozygous and 111 Homozygous Factor V Leiden Carriers: a Single-Center Cross-Sectional Study
Authors: Perez Botero Juliana , Ormsby Wayne D. , Ashrani Aneel A , McBane Robert D , Wysokinski Waldemar E , Patnaik Mrinal M. , Pruthi Rajiv K , Heit John A.
Published: October 2019

Background: While Factor V Leiden is associated with an increased risk of thrombosis, the magnitude of that risk among homozygous carriers is uncertain.

Methods: In a cross-sectional study of Mayo Clinic patients referred to the Special Coagulation Laboratory for a thrombophilia profile (n=379) over the 18-year period, 1996-2013, the prevalence of venous and arterial thrombosis was estimated for patients found to be Factor V Leiden carriers based on leukocyte genomic DNA genotyping. T tests and chi-square tests were used for continuous and categorical variables, respectively.

Results: Among homozygous (n=111) and heterozygous (n=268) carriers, the mean age at testing (50.3 and 50.5 years, respectively; p=0.9) and the proportion of females (50% and 54%, respectively; p=0.5) did not differ significantly. Of 29 asymptomatic carriers at the time of testing, 14 were homozygotes. Of the remaining 350 carriers, the mean age at first venous or arterial thrombosis (42 vs 43.8 years; p=0.5) among homozygous and heterozygous carriers, respectively, did not differ significantly. Thirty-five (33%) and 83 (31%) homozygous and heterozygous carriers, respectively, had at least one recurrent venous or arterial thrombosis (p=0.72). The prevalence of deep venous thrombosis (DVT) was 61% and 46% in homozygous and heterozygous carriers, respectively (p=0.008); the prevalence of pulmonary embolism was 18% and 20%, respectively (p=0.69). The prevalence of arterial thrombosis (mainly stroke and/or TIA) was higher in heterozygous (23%) compared to homozygous carriers (8%).

Conclusion: In the largest sample of homozygous Factor V Leiden carriers reported to date, thrombosis penetrance and phenotype did not appear to differ among homozygous vs. heterozygous carriers with the exception of a higher prevalence of DVT among homozygous carriers. These data should be interpreted with caution due to the potential for referral bias.

Disclosures

No relevant conflicts of interest to declare.
Calr Mutants Retroviral Mouse Models Lead to a Myeloproliferative Neoplasm Mimicking an Essential Thrombocythemia Progressing to a Myelofibrosis
Authors: Marty Caroline , Harini Nivarthi , Pecquet Christian , Chachoua Ilyas , Gryshkova Vitalina , Villeval Jean-Luc , Constantinescu Stefan N. , Kralovics Robert , Vainchenker William , Plo Isabelle
Published: October 2019

Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include Polycythemia Vera (PV), Essential Thrombocytemia (ET) and Primary Myelofibrosis (PMF). They are malignant homeopathies resulting from the transformation of a multipotent hematopoietic stem cell (HSC). The common mechanism of transformation is the constitutive activation of the cytokine receptor/JAK2 pathway that leads to the myeloproliferation. The acquired point mutation JAK2V617F is the most prevalent (95% of PV and 60% of ET or PMF). In addition, other mutations affecting the same signaling pathway have been described such as JAK2 exon 12 mutations, mutations of MPL affecting W515, and loss-of-function mutations of LNK and also mutations of c-Cbl in 3% of PMF. Recently, whole exome sequencing allowed identifying a new recurrent genetic abnormalities in the exon 9 of the calreticulin gene (CALR) in about 30% of ET and PMF patients. All CALR mutants induce a frameshift of the same alternative reading frame and generate a novel C-terminus tail.

To address the role of these new mutants in the pathophysiology of MPN, the goal of this study was to investigate the effect of the CALR mutant (del52 and ins5) expression by a retroviral mouse modeling.

For that purpose, we transduced bone marrow cells with retrovirus expressing either CALRdel52, CALRins5, CALRWT or CALRDexon9 and performed a transplantation in lethally irradiated recipient mice (10 mice / group), which were then followed over one year.

CALRdel52 expressing mice showed a rapid and strong increased in platelet counts (over 5 x106/mL) without any other changes in blood parameters during 6 months. In contrast, CALRins5 expressing mice presented platelet counts much lower than CALRdel52 but significantly higher than CALRWT or CALRDexon9 expressing mice. After 6 months, CALRdel52 expressing mice showed a decreased in platelets count associated with anemia and development of splenomegaly suggesting the progression to a myelofibrosis. Importantly, the disease was transplantable to secondary recipient for both CALRdel52 and CALRins5 mutants. The bone marrow and spleen were also analyzed over time. We observed a progressive increased in immature progenitors (SLAM cells) as well as a hypersensitivity of the megakaryocytic progenitors (CFU-MK) to thrombopoietin.

Altogether, these results demonstrate that CALR mutants are able and sufficient to induce a thrombocytosis progressing to myelofibrosis in retroviral mouse model, thus mimicking the natural history of MPN patients. It will offer a good in vivo model to investigate therapeutic approaches for CALR-positive MPN.

Disclosures

No relevant conflicts of interest to declare.
Microrna-374b Suppresses Proliferation and Promotes Apoptosis in T-Cell Lymphoblastic Lymphoma By Repressing Wnt-16 and AKT1 and Is a Promising Predictive Biomarker for Patients Survival
Authors: Cai Qingqing , Qian Dong , Chen Kailin , Xie Dan , Rao Huilan , Huang Huiqiang , Sun Xiaofei
Published: October 2019

Background: De-regulation of microRNA (miRNA) has been extensively investigated in both Hodgkin (HL) and non-Hodgkin lymphomas (NHL), however, little is known about the roles of miRNAs in T-cell lymphoblastic lymphoma (T-LBL). To understand the involvement of miRNAs in the development and treatment of (T-LBL), miRNA profiles were compared between tumor and corresponding non-tumor tissues.

Methods: miRCURY LNA array was used to generate miRNA expressing profile. Real-time quantitative PCR and immunohistochemistry (IHC) were applied to detect the expression of miR-374b, AKT1 and Wnt16 in T-LBL samples. Dual-luciferase reporter assay was conducted to confirm target associations of miR-374b. The tumor suppressive effect of miR-374b was determined by both in-vitro and in-vivo studies.

Results: The expression of 380 miRNAs was evaluated in five human T-LBL tissues and correspondence normal peripheral blood T-cells by microRNA microarrays. The down-regulation of miR-374b was frequently detected in primary T-LBL tissues, which was significantly associated with patients worse overall survival (P<0.001) and increasing risk of recurrence (P=0.012). Functional assays demonstrated that miR-374b could suppress T-NHL cells proliferation in vitro and in vivo. Furthermore, miR-374b could also sensitize cells to both serum starvation- and chemotherapeutic agent-induced apoptosis. Significantly, subsequent investigation characterized two AKT pathway associated molecules, AKT1 and Wnt16, as direct targets of miR-374b and account for the effect of miR-374b in T-NHL cells. Consistently, in tissues of our T-LBL patients, AKT1 and Wnt16 expression was inversely correlated with miR-374b levels, and could also be independent predictors of recurrence and survival of these patients.

Conclusions: Our data highlight the molecular etiology and clinical significance of miR-374b in T-LBL. Targeting miR-374b may represent a new therapeutic strategy to improve the therapy effect and survival for T-LBL patients.

Figure 1: Down-regulation of miR-374b is associated with poor prognosis of T-LBL Figure 1:. Down-regulation of miR-374b is associated with poor prognosis of T-LBL

Figure 2: ectopic expression of miR-374b suppress T-NHL cells proliferation and sensitize cells to both serum starvation- and chemotherapeutic agent-induced apoptosis Figure 2:. ectopic expression of miR-374b suppress T-NHL cells proliferation and sensitize cells to both serum starvation- and chemotherapeutic agent-induced apoptosis

Figure 3: Wnt16 and AKT1 are direct targets of miR-374b Figure 3:. Wnt16 and AKT1 are direct targets of miR-374b

Disclosures

No relevant conflicts of interest to declare.
STAT5b Gene Mutations and Their Association with a Unique Immunophenotype of CD4+CD56+TCRαβ Type LGL Leukemia
Authors: Tanahashi Takahiro , Matsuda Kazuyuki , Sekiguchi Nodoka , Nishina Sayaka , Senoo Noriko , Sakai Kaoko , Sakai Hitoshi , Ito Toshiro , Mitsui Takeki , Kwong Yok Lam , Ishida Fumihiro
Published: October 2019

Large granular lymphocyte leukemia (LGL L) includes T-cell LGL L and chronic lymphoproliferative disorders of NK cells (CLPD-NK) with indolent clinical course. STAT signaling system has been shown to play a crucial role in LGL L. We reported somatic mutations in the STAT3 gene in LGL L, which is associated with pure red cell aplasia among an Asian cohort. Since STAT5b mutations in LGL leukemia had also been discovered, we investigated mutations in STAT5b gene in Japanese patients with LGL L. Among 28 T-LGL L and 11 CLPD-NK, N642H and Y665F activating mutations in the SH2 domain of the STAT5b were found in 3 and 1 patient (pt)s, respectively, by direct sequencing, which was confirmed with the corresponding allele-specific (AS) PCR. The frequencies of STAT5b mutations in T-LGL L and CLPD-NK were 14.3% and 0%, respectively. Median age of the 4 pts was 76.5 years and mild neutropenia was recognized in 2pts, but no one was anemic. None of them showed apparent aggressive clinical courses, in contrast to previous reported cases (Rajala HLM et al, BLOOD 2013). Interestingly, STAT5b mutations were associated with a unique phenotype of LGL L with CD4+CD56+TCR αβ type (P = 0.0001). The mutations of STAT5b and STAT3 were mutually exclusive. In contrast to STAT3 mutation-positive patients, among whom significant part of the patients possessed STAT3-mutated subclones only detected with AS-PCR, no pt was identified to be positive for STAT5b mutation only with AS-PCR. We also investigated STAT5b mutations in 29 Chinese patients cohort with T cell LGL L by AS-PCR for N642H and Y665F, and found that no patient was positive for the mutations. Our results indicate that the STAT5b gene is mutated in T-LGL L, which would affect a specific immunophenotype of LGL. STAT5b and STAT3 mutations have significant but distinct contributions to the pathobiology of LGL L and STAT5b-mutated LGL L may consist a unique subtype of LGL L.

Disclosures

No relevant conflicts of interest to declare.
Diffuse Large B Cell Lymphoma (DLBCL) infiltrating T Cells Display an Activated and Exhausted Status and Are Inhibited By Ligands of Cosignaling Receptors Including PD-L1, PD-L2 and CD80 Expressed By Most DLBCL in Situ
Authors: Olive Daniel , Le Kieu Suong , Roussel Mikael , Broussais Florence , Azzaoui Imane , Rossille Delphine , Bouabdallah Reda , Fest Thierry , Xerri Luc , Lamy Thierry , Pangault Céline
Published: October 2019

The regulation of immune response through cosignaling receptors is the major mechanism that prevent normal responses against peripheral tissues but that is used by tumor to hijack the autologous immune responses. This therapy are currently among the most promising adjuvant therapy in cancers including lymphomas. In addition sPDL1 is an important marker of patient's prognosis (Rossille et al., Leukemia 2014)

Our objective was the study of the expression of ligands by activating receptors on diffuse large B cell lymphoma (DLBCL) in comparison to Follicular Lymphoma (FL), Hodgkin Lymphoma (HL) and hyperplasia and to investigate the status of the infiltrating immune system as well as the function of the sorted autologous interaction of T cells against autologous DLBCL

Methods: We have dissociated fresh tumor B cell samples ( FL= 13, HD= 15, DLBCL = 15) and analysed the immune microenvironment and DLBCL by flow cytometry using mAbs recognizing cosignaling molecules of 15 samples. In a second step we have sorted the T cells and autologous DLBCL and performed cocultures with the coincubation with antagonist PD1 (PD1.3.1) PDL1 (PDL1.3.1) PDL2 (PDL2.1), Tim3 and CD80 mAbs. T cell stimulation was performed for 5 days. We have previously described the proprietary antagonist PD1, PDL1, PDL2 and CD80 used (Serriari et al., J. Immunol., 2010; Ghiotto et al. , Int.Immunol., 2010)

Results: The immune cells including T cells present within the DLBCL microenvironment are characterized by a highly activated /exhausted status as evidenced by the expression of PD-1, PDL1, PDL2, CD80 and Tim3 Figure 1) . Importantly most of these receptors are involved in negative signals provided their ligand is expressed on their cognate target cell. PDL1, PDL2 and CD80 are both present on DLBCL with some degree of heterogeneity among tumors and surprisingly PD1. Autologous T cell and lymphoma cells were sorted and autologous responses such as proliferation and cytokine production were analysed after in vitro culture without or with antagonist mAbs directed against cosignaling receptors. We could evidence for the first time to our knowledge that blockade of PD1 and CD80 pathways induced /restored the autologous response of infiltrating T cells.

Conclusions: These data demonstrate that a subgroup of DLBCL patients might benefit from treatment with checkpoint blockade mAbs. A major objective is to understand the molecular basis of DLBCL heterogeneity that currently investigated.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Endogenous TGF-beta1 Mediated Osteogenic Impairment of Medullar Mesenchymal Stromal Cells in Primary Myelofibrosis
Authors: Martinaud Christophe , Desterke Christophe , Konopacki Johanna , Pieri Lisa , Golub Rachel , Schmutz Sandrine , Anginot Adrienne , Guerton Bernadette , Rochet Nathalie , Albanese Patricia , Henault Emilie , De Revel Thierry , Bourgeade Marie-Françoise , Alessandro Vanucchi , Lataillade Jean-Jacques , Le Bousse-Kerdilès Marie-Caroline
Published: October 2019

Primary myelofibrosis (PMF) is myeloproliferative neoplasm characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in bone marrow and spleen. Mesenchymal stromal cells (MSCs) are reported to play a pivotal role in fibrosis and stromal changes are considered as a reactive counterpart of the cytokine production by clonal hematopoietic cells. The present study shows that MSCs from patients demonstrate functional abnormalities that are unexpectedly maintained ex-vivo, in culture.

Material and Methods: we studied MSCs and bone marrow sections from PMF patients (n=12) as compared to healthy donors (HDs) (n=6). We tested their proliferation, immunophenotype, hematopoiesis supporting capacities, differentiation abilities, in-vivo osteogenic assays, and performed secretome and transcriptome analysis.

Results: We found that PMF-MSCs exhibit similar proliferative capacity and long-term hematopoiesis supporting abilities as compare to healthy donors. They overproduce interleukin 6, VEGF, RANTES, PDGF, BMP-2 and surprisingly TGF-beta1. MSCs from fibrotic PMF patients express high levels of glycosaminoglycans. Adipocytes and chondrocytes differentiation abilities were not different as compared to HDs but PMF-MSCs exhibit an increased in vitro potential. Implementation on scaffold in nude mice confirmed, in vivo, this increased osteogenic potential. We then looked into gene expression and discovered that PMF-MSCs show an original transcriptome signature related to osteogenic lineage and TGF-beta1. Indeed, osteogenic genes such as Runx2, Dlx5, Twist1, Noggin, Sclerostin, GDF5 and Serpine1 are deregulated and suggest a potential osteoprogenitor priming of PMF-MSCs. These molecular results also advocated for a TGF-beta1 impregnation that prompted us to study its impact on PMF-MSCs osteogenic differentiation. First, we then showed that Smad2 is intrinsically over-activated in PMF-MSC and that stimulation by TGF-beta1 is associated with an increase phospho-Smad2 level and an enhancement of bone master gene regulator Runx2 expression. Then, we inhibited TGF-beta1 pathway by by SB-431542 and evidenced a specific behavior of osteogenic MSCs differentiation in patients, suggesting involvement of TGF-beta1 in osteogenic impairment.

Conclusion: Altogether, our results identify a signature of PMF-MSCs and suggest that they participate in PMF osteogenic dysregulation independently from in vivo local stimulation by clonal hematopoietic cells

Disclosures

No relevant conflicts of interest to declare.
NEK2 Suppresses Cellular Senescence through Regulation of the β-Catenin/CCND2/RB Axis in Multiple Myeloma
Authors: Yang Ye , Tricot Guido J. , Jiliang Xia , Gu Zhimin , Xu Hongwei , Hao Mu , Zhan Fenghuang
Published: October 2019

Background: Multiple myeloma (MM) is a B-cell malignancy characterized by complex cytogenetic abnormalities and drug-resistance. We have previously shown that increased NEK2 enhances MM cell growth, survival and drug-resistance in vitro and in vivo. In this study, we explore whether high NEK2 is involved in overcoming cellular senescence, a state of irreversible proliferation arrest.

Materials and Methods: MM cell lines (ARP1, OPM2, OCI-MY5, and KMS28PE) with either over-expressed or knocked-down (shRNA) NEK2 (NEK2 OE or KD) by a lentiviral delivery system were used in this study. Cell senescence status was evaluated by staining SA β-galactosidase (SA β-gal). Flow cytometry was performed to examine cell cycle state in NEK2-KD or NEK2-OE MM cells treated with doxorubicin (30nM) for 48 hrs. Mouse embronic fibroblasts (MEFs) derived from NEK2-transgenic mice and wild type control mice were also used to verify the role of NEK2 in suppressing senescence and to elucidate signaling pathways related to NEK2 anti-senescence. Real-time PCR, immunofluorescence, co-immunoprecipitation (Co-IP) assay and western blot were employed for NEK2 mechanistic studies.

Results: NEK2-KD induced MM cellular senescence and cell cycle arrest, while NEK2 OE decreased doxorubicin-induced senescence and cell cycle arrest. Also, MEFs derived from NEK2 transgenic mice showed suppressed cell senescence. Mechanistic studies showed that NEK2 stabilized β-catenin and prevented degradation by the ubiquitin/proteasome pathway in MM cells. Intriguingly, immunofluorescence revealed that NEK2 co-located with β-catenin in the cell nucleus in NEK2 transgenic MEFs. Importantly, increased nuclear β-catenin up-regulated CCND2 expression, (but not CCND1 and CCND3) and phosphorylated RB in NEK2 over-expressed MM cells or NEK2 transgenic MEFs. Furthermore, LiCl, an inhibitor of GSK3β, was used to restore the activity of β-catenin in NEK2-KD cells. LiCl significantly increased CCND2 expression and blocked NEK2-shRNA induced cellular senescence in MM cells.

Conclusion: NEK2 prevented MM cell senescence. This may play an important role in NEK2 induced MM cell proliferation, survival and drug resistance. Prevention of senescence by NEK2 depended on its interaction with β-catenin. Increased β-catenin activity, caused by high NEK2 resulted in up-regulation of CCND2 and hyper-phosphorylation of RB.

Disclosures

No relevant conflicts of interest to declare.
Re-Educating Myeloma Associated Macrophages with Lenalidomide
Authors: Bruns Heiko , Gehlen Hanna , Nolting Jens , Pasemann Shirin , Brossart Peter , Mackensen Andreas , Gerbitz Armin
Published: October 2019

Introduction: The bone marrow niche plays a critical role in determining the fate of malignant plasma cells in multiple myeloma (MM). Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages can directly kill tumor cells and participate in antitumor immune responses as effector cells. Moreover, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies. Lenalidomide, an immunomodulatory drug (IMiD®) is used for the treatment of MM, also in the combination with therapeutic antibodies. Lenalidomide is thought to target the stromal support, but its precise influence on the phenotype or the effector functions of macrophages is still unclear.

Methods: To investigate the effect of lenalidomide on the interaction between macrophages and malignant plasma cells in vitro, we coincubated lenalidomide pretreated macrophages with several MM cell lines, and analysed the viability, proliferation and phenotype. For in vivo studies we utilized 5TMM mice, a suitable animal model for MM. Animals were treated with lenalidomide (50 mg/kg 5days/week) for 3 weeks, and the effector functions and phenotype of isolated bone marrow macrophages were analyzed. In addition, macrophages in the bone marrow of MM patients treated with lenalidomide were characterized by immunohistochemistry and flow cytometry.

Results: We showed, that infiltrating macrophages in the bone marrow of MM patients display an anti-inflammatory M2-like phenotype characterized by the expression of surface marker CD163, CD206, PD-L1 and cytokine/chemokine secretion (e.g. IL10, CXCL10, APRIL, BAFF and RANKL). Incubation of macrophages with lenalidomide in vitro, substantially changed their transcriptional program (e.g. downregulation of IRF4 and upregulation of IRF5) and their phenotype (e.g. downregulation of the surfaces marker CD163, CD206, and upregulation of CD16, CD64, CD40 and CD86). Furthermore, we show that lenalidomide treatment decreases the expression of RANKL, BAFF and APRIL, while tumoricidal effector molecules (e.g. TRAIL, cathelicidine, Granzyme B) were increased. When lenalidomide treated macrophages were cocultured with MM cells significant cytotoxicity was detected, for all MM cell lines tested. In contrast, untreated macrophages promote tumor growth and viability of MM cells.

Conclusion: Lenalidomide in vitro influences macrophages by reverting an anti-inflammatory M2 like profile to a more immunogenic phenotype. In addition it impacts on the support function by decreasing the secretion of important growth factors for B-cells. Similar results were observed in first in vivo studies. Taken together our results imply that lenalidomide interrupts an important stromal cell function thereby influencing survival of MM cells.

Disclosures

No relevant conflicts of interest to declare.
Lenalidomide Immunomodulation with an Allogeneic Myeloma GVAX in a Near Complete Remission Induces Durable Clinical Remissions
Authors: Noonan Kimberly , Huff Carol Ann , Sidorski Amy , Ferguson Anna , Rudraraju Lakshmi , Casildo Andrea , Borrello Ivan
Published: October 2019

Background: Increasing evidence suggests that the depth of response in myeloma (MM) correlates with delayed time to progression1.We have previously shown that lenalidomide (Len) can augment vaccine efficacy to the pneumococcal conjugate vaccine (PCV), Prevnar2.We extend those observations to examine whether vaccinating patients on Len in a near complete remission (nCR) (negative M-spike, IFE positive) could further deepen the clinical response and generate measurable myeloma specific immunity in a small Phase II study.

Design:Patients needed to be on a Len-containing regimen and needed to be in one of the following categories for enrollment: 1) in a stable nCR for at least 4 months; 2) converting from IFE negative to IFE positive; or 3) showed signs of early relapse from a nCR to an M-spike <0.3g/dL. Patients would be continued on single agent Len and receive 4 vaccinations consisting of two allogeneic MM lines, H929, U266 admixed with K562 transduced to express GM-CSF as well as PCV. Patients received 3 monthly vaccines and a boost at 6 months.

Results: To date 32 patients have been screened: 12 had relapsed and 5 entered into an IFE negative CR during the 4 month observation period; 11 have been enrolled. Median follow-up for the study is 13.9 months. Of the vaccinated patients, category 1: 4 patients: 3 are in a true CR (IFE-),1 lost the original clone and is still in a nCR. Category 2: 2 patients both achieved a true CR. Category 3: 5 patients: 2 stable disease, 3 progressive disease. The overall true CR rate is 54%.of the patients that achieved a CR with a median follow-up of 17.1 months. Laboratory analysis showed that the patients achieving a CR had more central memory CD8 T cells at baseline in the BM and blood, a greater activation phenotype in CD8 cells in the BM and greater tumor specific IFNg production. Interestingly, LAG-3, TIM-3 and PD-1 on CD8 T cells were more expressed in the BM of CR patients.

Conclusions: Vaccination in combination with Len appears to durably deepen the clinical response in patients in a nCR whereas patients with early relapse appear less likely to respond. The deepening the clinical response correlates with the development of myeloma-specific immunity. Vaccination of MM patients in nCR in combination with Len shows promising early clinical activity that warrants further investigation as an approach to maintaining durable clinical remissions.

1.Chanan-Khan, A.A., and S. Giralt. 2010. Importance of achieving a complete response in multiplemyeloma, and the impact of novel agents. J Clin Oncol 28:2612-2624.

2. Noonan, K., L. Rudraraju, A. Ferguson, A. Emerling, M.F. Pasetti, C.A. Huff, and I. Borrello. 2012. Lenalidomide-induced immunomodulation in multiple myeloma: impact on vaccines and antitumor responses. Clin Cancer Res 18:1426-1434.

Disclosures

Noonan: Celgene: Speakers Bureau. Sidorski:Celgene: Speakers Bureau.
Maintenance of Respiration Capacity Is an Early Predictor of Drug Resistance in Cancer Cells
Authors: Brendel Cornelia A , Henkenius Katharina , Maerken Melanie , Kaiser Tom , Greif Larissa , Metzelder Stephan , Rehberger Miriam , Neubauer Andreas
Published: October 2019

Inherent or acquired drug resistance is the major obstacle in cancer therapy. An individual prediction of response to cancer therapy would be highly appreciated. It has been reported that the mitochondrial function of cancer cells correlates with the treatment response in different cancer entities (Chonghaile et al., Science 2011 and Vo et al., Cell 2012).

However, as the method of BCL-2 peptide priming measurement (“BH3 profiling”) seemed to be rather complicated and error-prone in our hand we sought to determine mitochondrial function by analyzing cellular respiration. We established three different drug resistant human cancer cell lines (MV4-11, HL-60 and NCl-H82) by continuous exposure to Sorafenib, Cytarabine (Ara-C) or Etoposid (VP-16) respectively. Cellular respiration was measured as oxygen consumption rate (OCR) 24h after drug exposure employing the XF96 extracellular Flux Analyzer. Viability, reduction equivalents NADH/NADPH and mitochondrial membrane potential of the cells were concomitantly determined by flow cytometry via 4’, 6-diamidino-2-phenylindole (DAPI) exclusion, (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Tetramethylrhodamine ethyl ester (TMRE) staining with flow cytometric analysis, respectively.

All resistant cancer cells maintained their respiration capacity upon exposure to tyrosine kinase inhibitors, chemotherapeutics or topoisomerase inhibitors, whereas the respiration of drug sensitive cancer cells decreased significantly. Moreover, resistant MV4-11 and HL-60 cells exhibited a metabolic shift towards glycolysis after drug treatment. In contrast sensitive cancer cells showed a decline in respiration and glycolysis in a dose dependent manner. Decrease of respiration was evident at 24h post treatment, while the onset of apoptosis was measurable 48-120h later. Reduction equivalents NADH/NADPH but not mitochondrial membrane potential were diminished in sensitive cells after 24h drug exposure, but dose dependency was more distinct with OCR analysis. The increase of glycolysis in resistant cell lines was also not detectable with the MTT assay.

Our data indicate cellular metabolism and in particular respiration as an early, sensitive and reliable surrogate parameter of drug sensitivity in viable cells that might therefore be applicable in order to determine drug responsiveness in primary cancer cells in a prospective manner.

Disclosures

No relevant conflicts of interest to declare.
Targeting C-Myc Promoter: Helquats As Novel G-Quadruplex Stabilizing Ligands
Authors: Kuzmova Erika , Kozak Jaroslav , Komarkova Veronika , Pytlik Robert , Teply Filip , Miroslav Hajek
Published: October 2019

Influencing stability of G-quadruplexes that can be formed in specific guanine-rich sequences has emerged as a promising route to anticancer drug design in recent years. These sequences are highly prevalent in human genome and occur in the telomeres as well as in promoters of certain genes upregulated in cancer (e.g. c-Myc, K-ras, VEGF, bcl-2, c-kit, or HIF-1α). Functional role of these G-quadruplex structures has been tested using G-quadruplex-stabilizing ligands in cell-based assays, resulting in suppression of transcription of oncogenes. As c-Myc deregulation is found in many types of human leukemia and lymphoma and its overexpression is often associated with disease progression, we chose it as our primary target.

We designed a library of helical structures with cationic aromatic moieties called helquats to find a novel class of G-quadruplex specific ligands. Their non-planar structure minimizes potential of intercalation into DNA duplex. To screen a library of about 1500 compounds synthesized in-house and find ligands that stabilize c-Myc G-quadruplex at low micromolar concentrations, we used G-quadruplex forming sequence NHE III1 of c-Myc promoter in various biophysical and biochemical experiments such as FRET (Förster resonance energy transfer) melting experiments, CD spectroscopy, and DNA polymerase stop assay. We tested promising candidates for reducing c-Myc mRNA and protein expression in several leukemic cell lines and in reporter assay using an experimental vector with firefly luciferase under control of c-Myc promoter region containing either wild-type or mutated G-quadruplex NHE III1 sequence. The most potent helquats showed more than 50 % inhibition of c-Myc mRNA and protein expression (at 25 μM, after 24 hours) in HL-60, Ramos, and U937 cell lines overexpressing c-Myc. On the other hand, no such an effect of helquats was observed in CA46 Burkitt lymphoma cells in which NHE III1 element has been lost during the translocation. Similar results as with cell lines were obtained in primary cells with or without c-Myc translocation.

Our results provide the foundation for the development of new helquat derivatives as effective inhibitors of c-Myc expression and as potential anticancer agents in hematooncology.

This study was supported by IOCB, AS CR, v.v.i. (RVO: 61388963), Project InterBioMed LO1302 from Ministry of Education of the Czech Republic and PRVOUK-27/LF1/1

Disclosures

No relevant conflicts of interest to declare.
Clinical Features and Outcomes of Iron Deficiency Anemia in Patients with Gastrectomy
Authors: Ryu Hyewon , Jo Deog-Yeon , Lee Kyu-Seop , Song Ik Chan , Choi Yoon Seok , Moon Ji-Young , Lee Myung-won
Published: October 2019

Background: Iron deficiency anemia (IDA) is common in patients with partial or total gastrectomy; however, clinical features and appropriate therapy in these patients have not been well described. We compared clinical features and outcomes in IDA patients with and without gastrectomy.

Methods: We retrospectively analyzed clinical features at the time of diagnosis and response to iron therapy in IDA patients with partial or total gastrectomy (gastrectomy group) and in age- and gender-matched IDA patients without gastrectomy (control group) who were diagnosed between 2002 and 2010 at Chungnam National University Hospital.

Results: A total of 2,072 patients were diagnosed with IDA during the study period. Thirty-seven and 40 patients were enrolled in the gastrectomy and control groups, respectively. The median ages were 58.8 years (range, 19–84 years) and 52.7 years (range, 25–84 years), respectively. At initial presentation, the hemoglobin levels, mean corpuscular volume (MCV), mean corpuscular hemoglobin levels, mean corpuscular hemoglobin concentration, serum iron levels, total iron binding capacity, ferritin levels as well as endoscopic findings did not differ between the two groups. After 2 weeks, 6 weeks, and 3 months of oral iron therapy, hemoglobin levels were significantly lower in the gastrectomy group than in the control group (8.3 vs. 9.6 g/dL, P=0.012; 9.7 vs. 12.0 g/dL, P=0.000; 11.9 vs. 13.3 g/dL, P=0.016, respectively). After 2 weeks and 6 weeks of treatment, the increase in the hemoglobin level and that in the MCV were significantly slower in the gastrectomy group than in the control group (hemoglobin: 0.6 vs. 2.3 g/dL, P=0.000; 2.1 vs. 4.7 g/dL, P=0.000, respectively) (MCV: 2.6 vs. 6.2 fL, P=0.005; 7.2 vs. 12.7 fL, P=0.008). Parenteral iron was required in nine (23.4%) patients in the gastrectomy group, but not in the control group. The follow-up duration was much longer in the gastrectomy group (23.2 vs. 5.2 months, P=0.008), and 11 (29.7%) patients in this group needed long-term maintenance iron therapy. To normalize hemoglobin levels, longer treatment was required in the gastrectomy group (0.8 vs. 2.8 months, respectively; P=0.013).

Conclusion: The response to oral iron therapy in IDA patients with gastrectomy is slower than in those without gastrectomy. Parenteral iron therapy is needed in one-fourth of these patients to normalize hemoglobin levels.

Disclosures

No relevant conflicts of interest to declare.
ASPP2 Attenuation Is an Early Step in Leukemogenesis Facilitating Acquisition of Mutations
Authors: Kampa-Schittenhelm Kerstin M , Akmut Figen , Illing Barbara , Lopez Charles , Schittenhelm Marcus M
Published: October 2019

Inactivation of the p53 pathway is an universal event in human cancers. As p53 mutations are rare in de novo acute leukemias, the p53-pathway must be inactivated by other mechanisms instead. The Apoptosis stimulating Protein of p53-2 (ASPP2) is a haploinsufficient tumor suppressor, which we have shown to be attenuated in acute leukemia. We now provide evidence that ASPP2 regulates pathways involved in early hematopoiesis, and attenuated expression perturbs malignant transformation.

Gene-chip microarrays were performed on ASPP2+/+ versus +/- mouse embryonic fibroblasts (MEFs) (-/- knockouts are not viable) and pathway analysis was performed using Affimetrix software. ASPP2 expression was silenced in IL3-dependent Ba/F3 pro-B cells and leukemia cell lines using standard siRNA protocols. Cellular proliferation was assessed using XTT-based assays. To evaluate for malignant transformation, long term cell cultures with stably retrovirally silenced Ba/F3 cells (+/- stress inforcement using gamma irradiation, 4x 5Gy) were employed. IL3 weaning was performed as an indicator of autoactivated cellular proliferation, and analysis of chromosomal defects or gene mutations were performed.

Microarray mRNA analysis revealed that attenuated ASPP2 expression resulted in significant alterations of pathways involved in mediating cellular growth, proliferation and tissue differentiation. Subanalysis proofed involvement of genes playing a major role in embryogenesis, the development of the hematopoietic system and leukemogenesis (homeobox (HOX) family members, NOTCH, BCL, IRF7 and EGR1). Population doubling times were significantly increased in ASPP2+/- MEFs compared to the parental cells. Modeling ASPP2 attenuation via specific siRNA knockdown in leukemia cell lines (K562 and Kasumi1) resulted in significantly increased cellular proliferation. Similarly, ASPP2-interference in pro-B Ba/F3 cells lead to perturbed proliferation and an increase of polyploid cells with a dramatic change in morphology with megaloid cells, indicating mitotic failure. This effect was even more pronounced after stress induction with daunorubicin. Lentiviral shRNA-ASPP2 stable knock-out was performed in Ba/F3 cells to follow cells in long term cultures for malignant transformation. Again, we confirmed signs of mitotic failure as observed in the siRNA approach. Even more, gamma irradiated ASPP2-lacking strains were successfully IL3 weaned, which indicates autoactivated cellular proliferation. A screen for acquired oncogenic mutations was performed and analyses are ongoing.

In summary, we provide evidence that ASPP2 has a pivotal role in early hematopoiesis, and loss of ASPP2 is a driver mechanism to fuel leukemogenesis.

Disclosures

No relevant conflicts of interest to declare.