bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel ICAM-1/CD54 Antibody Identified By Phage Display with Potent Pre-Clinical Anti-Myeloma Activity
Authors: Klausz Katja , Burger Renate , Kellner Christian , Peipp Matthias , Gramatzki Martin
Published: October 2019

Introduction: Despite novel agents in multiple myeloma (MM) a substantial number of patients is still in need of better disease control. Monoclonal antibodies provide therapeutic options in other tumor entities but in MM no antibody has been approved yet. Two antibodies currently evaluated namely Daratumumab (CD38) and Elotuzumab (CD319), are ahead and achieved phase 3 clinical trials, but other antigens on myeloma cells could also be promising targets. Here, a cell-based phage display screening strategy is presented that identified a novel intercellular adhesion molecule-1 (ICAM-1)/CD54 antibody suitable for myeloma immunotherapy.

Methods: Synthetic human single-chain fragment variable (scFv) phage display libraries were pre-absorbed with granulocytes and T lymphocytes of healthy individuals and subsequently panned with plasma cell lines INA-6 and JK-6L. Phage binding characteristics were evaluated by cellular ELISA and flow cytometry. The coding sequence of the leading candidate MSH-TP15 was used to generate scFv-Fc fusion proteins and fully human IgG1 antibodies with wild type or modified Fc domains for functional in vitro and in vivo testing.

Results: Panning of human phage display libraries with a newly established subtractive cellular screening approach resulted in phages with preferential binding to MM cells. Based on its reactivity with MM cell lines and patient-derived CD138+ malignant plasma cells from bone marrow, pleural effusion and peripheral blood, the leading candidate MSH-TP15 was selected. Notably, MSH-TP15 showed no or only marginal binding to B and T lymphocytes, NK cells, monocytes and granulocytes of healthy individuals and MM patients. Recent studies identified ICAM-1/CD54 as the antigen recognized by MSH-TP15. The antibody alone was not capable to significantly inhibit growth of plasma cell lines in vitro, however induced antibody-dependent cell-mediated cytotoxicity (ADCC), an important killing mechanism of many therapeutic antibodies. An Fc-engineered antibody variant of MSH-TP15 triggered dose-dependent ADCC against INA-6, L363, RPMI-8226, U266 and MM1.S plasma cell lines and freshly isolated tumor cells using blood mononuclear cells and NK cells of healthy donors as effector cells. In contrast, complement-dependent cytotoxicity was not induced. Importantly, in vivo studies revealed that MSH-TP15 completely prevented tumor engraftment in the INA-6 myeloma xenograft model.

Conclusions: The cell-based screening of phage display libraries led to the identification of a novel ICAM-1/CD54 antibody that is effective in antibody-mediated cellular cytotoxicity of MM cells in vitro and completely prevented myeloma tumor engraftment in vivo. The relevant mechanisms of action of MSH-TP15 will be defined with different antibody variants and in vivo models to further evaluate its potential for an antibody-based treatment approach of MM.

Disclosures

No relevant conflicts of interest to declare.
Novel Series of ALK2 Inhibitors That Alter Hepcidin Expression As Potential Therapeutics for Anemia of Chronic Disease
Authors: Kim Wontak , Maughan Kyle S. , Soh Katherine K. , Bearss Jeremiah J. , Bahr Brigham L. , Bearss David J. , Warner Steven L.
Published: October 2019

Anemia of chronic disease (ACD) is an inflammatory cytokine driven disease characterized by hypoferremia despite adequate iron stores. This is largely due to hepcidin, a master regulator of iron homeostasis, which blocks enterocytes from absorbing iron and preventing iron release from macrophages by binding to ferroportin. It is known that bone morphogenetic proteins (BMP) up-regulate hepcidin by activating the SMAD signaling pathway through the activin-like kinase receptor 2 (ALK2). Therefore, ALK2 has emerged as a potential therapeutic target to modulate hepcidin levels and treat ACD.

We have developed a novel series of small molecule ALK2 inhibitors with promising activity in preclinical models of ACD. Using well-established cell-based and animal models of hepcidin signaling and anemia, we optimized and validated the activity of the most promising preclinical lead candidates. These compounds demonstrate significant activity in downregulating hepcidin expression in BMP-induced cell culture studies at concentrations of 100 nM or lower. Importantly, this hepcidin lowering activity was observed at concentrations that exhibited no cytotoxicity suggesting the compounds have a clean selectivity profile. The compounds also demonstrated remarkable activity in animal models of anemia, including an acute model induced by the administration of turpentine oil and a more chronic model induced by tumor formation and growth. Treatment with the lead candidates completely reversed the induction of hepcidin expression in these models and also decreased the symptoms of anemia as measured by serum iron and red blood cell levels. From these data, we have nominated a candidate to advance into IND-enabling studies that has favorable drug-like properties. We anticipate a clinical development strategy that focuses on anemia of cancer with subsequent expansion into anemia associated more broadly with other inflammatory and chronic diseases

Disclosures

Kim: Tolero Pharmaceuticals: Employment. Maughan:Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Bahr:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.
An Inflammatory Pathway Mediating Rapid Hepcidin-Independent Hypoferremia
Authors: Muckenthaler Martina U. , Guida Claudia , Altamura Sandro , Klein Felix A. , Galy Bruno , Boutros Michael , Ulmer Arthur J. , Hentze Matthias W.
Published: October 2019

Hypoferremia represents an innate immune response to infection and inflammation sequestering iron from pathogens. The iron-hormone hepcidin is induced by such stimuli, causing degradation of the iron exporter ferroportin (Fpn) and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here we report the discovery of a fast, hepcidin-independent hypoferremia pathway. Stimulation of the toll-like receptor (TLR) 2 and TLR6 triggers profound decreases in Fpn mRNA and protein expression in bone marrow-derived macrophages, liver and spleen of mice without changing hepcidin expression. Furthermore, C326S Fpn knock-in mice with a disrupted hepcidin/Fpn regulatory circuitry respond to injection of the TLR2/6 ligand FSL1 by Fpn down regulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia, uncovering a rapid and potent inflammatory response pathway.

Disclosures

No relevant conflicts of interest to declare.
Predisposition to Burkitt Lymphoma in Williams-Beuren Syndrome
Authors: Guenat David , Rizzari Carmelo , Lundin Catarina , Borg Christophe , Soulier Jean , Rohrlich Pierre-Simon
Published: October 2019

Williams-Beuren Syndrome (WBS) is a multisystem disorder caused by a 1.5 Mb hemizygous deletion at chromosome 7q11.23. Here, we report two novel cases of Burkitt lymphoma in WBS children. With the addition of these latest two case reports the number of pediatric cancer reported in WBS has reached 11 cases. Strikingly, 8 (73%) of them were Burkitt lymphoma. In addition, a sporadic Burkitt lymphoma in a third child without WBS but carrying a somatic deletion of the WBS critical region was investigated. Array-based Comparative Genomic Hybridization and Next Generation Sequencing were used to analyze the DNA isolated from normal tissues and lymphoma cells of the 3 patients. Microsatellite instability was also investigated. The constitutional hemizygous deletion observed in the normal DNA of the 2 WBS patients corresponded to the typical WBS critical region and there was no evidence of homozygous deletion or mutation at 7q11.23 in the tumor DNA. Remarkably, the somatic deletion observed in the sporadic NHL was similar to the one observed in WBS patients. Microsatellites were stable in the 3 patients. A number of genes mapping to the WBS critical region are involved in DNA repair (PMS2L, BAZ1B, RFC2, GTF2I family genes). Haploinsufficiency of one or of a combination of these genes could be involved in the predisposition to Burkitt lymphoma in patients with WBS. More generally, this observation illustrates the putative role of genes located at 7q11.23 in lymphomagenesis, thus pointing to potentially new targets for anti-cancer therapies.

Disclosures

No relevant conflicts of interest to declare.
PARP Inhibitor Selectively Induces Cell Death in E2A-Hlf Positive Leukemia
Authors: Takagi Masatoshi , Piao Jiuhua , Kamiya Takahiro , Masutani Mitsuko , Mizutani Shuki
Published: October 2019

Background: Defects in homologous recombination repair (HRR) have long been known to contribute to genomic instability leading to tumor development. Poly (ADP-ribose) polymerase (PARP) exerts various cell biological effects, such as maintenance of genomic stability, energy metabolism and cell death. PARP is indispensable in DNA repair machinery, especially in base excision repair (BER). PARP inhibition convert DNA double strand breaks from DNA single strand breaks induced by alkylating agents. These DNA double strand breaks can be repaired by HRR. Therefore, PARP inhibitor induces synthetic lethality in HRR defective cancer cells. Such lethality was successfully shown in BRCA1 or 2 mutated breast cancers. However, only a limited study has been performed other than breast cancers. Some tumors including hematological malignancies are defective in HRR function leading to a possibility to be sensitized to PARP inhibitor.

Methods: Sensitivity to PARP inhibitor was screened using 28 leukemia cell lines. HRR activity was measured by DR-GFP HRR assay. Expression of proteins involves HRR was evaluated by cDNA microarray analysis and western blotting.

Results: E2A-HLF positive leukemia showed susceptibility to PARP inhibitor. This experiment suggests that expression of E2A-HLF chimeric messenger RNA sensitize the leukemic cell to PARP inhibitor. To elucidate whether E2A-HLF genuinely sensitize the cell for PARP inhibition, E2A-HLF was transduced into PARP inhibitor resistant Burkitt cell line, Daudi, using retrovirus. Compared with mock infected Daudi cell, E2A-HLF expressed Daudi cell showed increased sensitivity to PARP inhibitor. This experiment suggests that E2A-HLF expressed cell is defective HRR pathway. To test this hypothesis, HRR assay using DR-GFP construct was employed. HRR between the two nonfunctional GFP genes to generate a functional GFP gene can be triggered by transient transfection of the I-SceI expression vector, which introduces a DNA double-strand break (DSB) in one of the two GFP genes. HRR proficiency can be determined by the number of cells expressing the GFP protein. DR-GFP HRR assay exhibited defect of HRR function in E2A-HLF expressed cell. Interestingly, expression of BRCA1 was decreased in E2A-HLF transfected cell, which presumably link with decreased HRR activity.

Conclusions: Increased sensitivity to PARP inhibitor in E2A-HLF positive leukemia was caused by decreased HRR activity by E2A-HLF expression. PARP inhibitor will be a novel therapeutic approach for refractory leukemia, especially with E2A-HLF translocation. While PARP inhibitor monotherapy is an attractive proposition for treating such as HRR defective E2A-HLF expressed leukemia, combination of HRR inhibitor will be a universal strategy for various types of leukemia.

Disclosures

No relevant conflicts of interest to declare.
Egr-1, a Stress Response Transcription Factor and Myeloid Differentiation Primary Response Gene, Behaves As Tumor Suppressor in CML
Authors: Maifrede Silvia , Liebermann Dan , Hoffman Barbara
Published: October 2019

The transcription factor early growth response 1 (Egr-1) gene was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There’s evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with therapy derived MDS and AML; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). BCR-ABL driven leukemia (Chronic Myelogenous Leukemia [CML]) was chosen as a model system to investigate the role of Egr-1 as a tumor suppressor for leukemia. CML is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. To assess the effect of Egr-1 on BCR-ABL driven leukemia, lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-GFP retrovirus (bone marrow transplantation {BMT}). It was observed that loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Analysis of hematopoietic organs is consistent with loss of Egr-1 accelerating development of CML but not altering the nature of the disease. Cells expressing BCR-ABL that are null for Egr-1 exhibit increased survival and proliferation, which can account for some of the above observations. Substantial evidence is available that loss of Egr1 increases the leukemia initiating cell population, which can account for the more rapid development of disease. An increased population of lineage negative BM cells was observed in Egr-1-/- BCR-ABL recipient mice compared to animals transplanted with WT BCR-ABL BM. Serial BMT has shown that Egr-1-/- BCR-ABL BM has an increased leukemic burden compared to the WT counterpart. Furthermore, data from serial colony transfer experiments confirmed that loss of Egr-1 increases leukemia initiating cells. Preliminary studies on the effect of gain of Egr-1 function are consistent with the tumor suppressor behavior of Egr-1. These data as well as analysis of human CML samples will be presented. Further investigation could result in novel targets for diagnosis, prognosis, and targeted therapeutics, including strategies for activating Egr-1 expression that can be used to treat CML, as well as other leukemic diseases.

Disclosures

No relevant conflicts of interest to declare.
New Mutation in Chromosome 9 Associated with Familial Autosomal Recessive Macrothrombocytopenia
Authors: Revel-Vilk Shoshana , Shai Ela , Gashem Nivin , Hyam Esti , Spectre Galia , Elpeleg Orly , Varon David
Published: October 2019

We report a family with new autosomal recessive disorder associated with macrothrombocytopenia which is different from all reported hereditary macrothrombocytopenia.

Case report: The affected family members report mild bruising and epistaxis. The females’ family members report, in addition, severe menorrhagia and recurrent episodes of hemorrhagic corpus luteum. Two family members with macrothrombocytopenia and two without macrothrombocytopenia report hearing loss and cataracts.

Methods: The study protocol was approved by our local Ethics committee and written consent was obtained to prepare and store DNA. Platelet morphology, FACS analysis, protein analysis and genome-wide association studies were used to characterize the disease and locate the genetic abnormality.

Results: In a consanguineous family from Arab origin we identified 5 affected individuals (3 males and 2 females from three families) with platelet count ranging around 4-20 x109/L. Their shared grandparent died of post trauma bleeding (no data on his platelet count). Their parents have normal platelet count and platelet morphology. Blood film of effected family members show macrothrombocytopenia with occasional giant platelets. By flow cytometry CD41 and CD42b expressions were around 60% in effected family members and CD62 expression was around 20-30% in resting and thrombin-activated platelets (Figure 1). Immunoflurescence of platelets shows normal distribution of non-muscular mysoin IIA, GPIbIX, dense granules and lysosome markers Lamp-1, Lamp-2 and the dense granule CD63. Platelets diffusely stained for P-selectin and stained very low for VWF and thrombospondin. GPIIbIIIa staining revealed a ring distribution at the outer cell membrane (Prof. Andreas Greinacher, Greifswald, Germany). Transmission electron microscopy imaging shows abnormal distribution of platelet granules, with suspected protein accumulation in the cytoplasm (Figure 2). Protein analysis of platelet extracts by SDS-PAGE separation shows different band patterns in the effected individuals compared to controls (Figure 3). Mass spectrometry analysis of a representative band identified five candidate proteins that their over-expression may cause the defect in platelets organelles distribution and the defect of their function. Through whole genome analysis three large homogenous regions were found. To further narrow the search SNP analysis was added between two effected members. The search was narrowed to an area in chromosome 9p and centromere 9q with 201 candidate genes.

Discussion: We present a large consanguineous family with previously non reported autosomal recessive macrothrombocytopenia. The functional and morphological pattern show dual platelet population. Further work is warranted.

Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3.

Disclosures

No relevant conflicts of interest to declare.
Carboxypeptidase B2 Is Protective in a Mouse Model of Shiga Toxin-Induced Hemolytic Uremic Syndrome
Authors: Nishimura Toshihiko , Morser John , Shao Zhifei , Leung Lawrence L.
Published: October 2019

Hemolytic uremic syndrome (HUS) is characterized by a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. The most common cause of HUS is Shiga toxin (STX)-producing E. coli, and eculizumab, a monoclonal antibody against complement C5, has shown clinical efficacy in some patients. Carboxypeptidase B2 (CPB2) is a metalloprotease activated by the thrombin/thrombomodulin complex that inactivates a number of inflammatory mediators, including complement C3a, and C5a by removing their C-terminal arginine. We hypothesized that in a murine model of STX-induced HUS, Cpb2-/- mice would have exacerbated disease compared to wild type (WT) mice due to excessive C3a and/or C5a in the absence of CPB2. A mouse model of STX-induced HUS was established by giving STX and LPS toxins intraperitoneally. Cpb2-/- mice had worse survival than WT (37% survival vs. 87% at 48h, p=0.0156). At 48h, severe thrombocytopenia developed in both WT and Cpb2-/- mice (WT: 0.096x106/μL; Cpb2-/-: 0.054x106/μL) compared to controls (1.2x106/μL; p>0.0001 vs. either WT or Cpb2-/-), with Cpb2-/- mice showing worse thrombocytopenia. Renal insufficiency was worse in Cpb2-/- mice than WT mice (BUN at 48h: 85 mg/dL vs. 37 mg/dL, p=0.0074; creatinine: 1.33 mg/dL vs. 0.23 mg/dL; p=0.0112, for Cpb2-/- and WT mice respectively, compared with normal baseline BUN and creatinine of 19 mg/dL and 0.1 mg/dL). Cpb2-/- mice developed worse anemia than WT (hemoglobin 9.8 g/dL vs. 12.4 g/dL, p=0.001 in Cpb2-/- vs. WT mice respectively). At 48h, liver function was worse in Cpb2-/- mice than WT mice, while plasma LDH was increased in Cpb2-/- mice more than WT mice. Using a standardized health score, the Cpb2-/- mice were worse than WT mice at all time points. Thus this model recapitulates STX-induced HUS with the Cpb2-/- mice having worse disease than WT. If the animals were treated with STX alone, there were no deaths in either genotype at 48h and only 37.5% mortality in Cpb2-/- mice by 60h compared with no deaths in WT mice. BUN, creatinine, liver enzymes and LDH were increased in both genotypes treated with STX alone compared to untreated mice, but there was no significant difference between the genotypes. Treatment with LPS alone caused thrombocytopenia in both WT and Cpb2-/- mice and LDH, BUN and creatinine levels were higher in Cpb2-/- mice than in WT mice, but there was no death at 48h and no drop in hemoglobin. Thus while either STX alone or LPS alone caused pathological conditions in the mice, the typical triad of HUS was only present when STX and LPS were given in combination. The Cpb2-/- mice had worse disease than WT mice consistent with our hypothesis on the role of CPB2 in inactivating C3a and/or C5a in STX-induced HUS. The potential efficacy of C3a and/or C5a blockade and anti-thrombotic agents will be tested in this model.

Disclosures

No relevant conflicts of interest to declare.
Resistance to Thrombomodulin but Not to Activated Protein C in Cirrhotic Patients
Authors: Sinegre Thomas , Abergel Armand , Duron Cedric , Berger Marc G , Lebreton Aurélien
Published: October 2019

Introduction: Cirrhotic patients have an impairment of haemostasis. They are exposed to both thrombotic and bleeding events. Global coagulation tests as thrombin generation assays (TGA) could be informative to evaluate the coagulation step in this context. When the protein C (PC) pathway is sensibilised using thrombomodulin (TM), a « resistance » to the PC pathway appears, increasing with the severity of the hepatopathy. Nevertheless, patients with cirrhosis have a decrease protein C level which is probably involved in this « resistance ». The aim of this study was to compare the TGA with thrombomodulin or activated PC (aPC) to by-pass the PC defect.

Materials and Methods: Patients prospectively included were confirmed cirrhotic patients (prothrombin time <70% and/or liver dysmorphia and/or fibroscan> 20 kPa and/or histology and/or association of portal hypertension and liver failure). Patients were exclusively male, free of hepatocellular carcinoma and were not anticoagulated. Patients with on-going infection or inflammatory complication were excluded. None of them had a thromboembolic event or a familial history of thromboembolism.

TGA were performed in the presence/absence of TM and in the presence/absence of aPC, with previously determined concentrations inhibiting 50% of endogeneous thrombin potential (ETP) of healthy plasmas. Results were expressed as ratios with/without TM or with/without aPC. Statistical analysis was based on Kruskal-Wallis test and Dunn's post test. All tests were performed in triplicate. The study met all ethical autorizations.

Results: We enrolled 15 Child-Pugh A, patients 11 Child-Pugh B, 10 Child-Pugh C and 14 healthy controls. Comparatively to controls, PC, protein S (PS) and FVIII levels were significantly decreased in cirrhotic patients. ETP ratio with TM were statistically different between controls vs Child-Pugh B (p<0.05) and controls vs Child-Pugh C (p<0.01). Peak height ratio with TM were statistically different between controls vs Child-Pugh A (p<0.05), controls vs Child-Pugh B (p<0.01) and controls vs Child-Pugh C (p<0.01). All these significant differences disappear when we used aPC instead of TM, suggesting that the « resistance » to the soluble TM is due, at least partially, to the acquired deficiency in PC.

Discussion and Conclusions: The main finding of this study is that the acquired protein C deficiency actively participate to the « resistance » to soluble thrombomodulin in cirrhotic patients. These data are in line with studies of the Tripodi group who have also demonstrated a participation of the protein C deficiency in the resistance to thrombomodulin and Protac. Further studies are needed to evaluate the association between this biological phenotype and thrombotic events.

Disclosures

No relevant conflicts of interest to declare.
Notch Signaling By Either Notch1 or Notch2 Mediates Expansion of AGM-Derived Long-Term HSC Populations in Vitro
Authors: Hadland Brandon K , Varnum-Finney Barbara , Moon Randall T , Poulos Michael Gustave , Butler Jason M. , Rafii Shahin , Bernstein Irwin D.
Published: October 2019

Long-term, adult-engrafting hematopoietic stem cells (HSC) first emerge from hemogenic endothelial (HE) precursors in the context of embryonic arterial vessels such as the dorsal aorta of the AGM (Aorta-Gonad-Mesonephros region), a process which requires Notch1 receptor signaling. However, a possible subsequent role for Notch receptor activation during the unique period of substantial HSC expansion in embryonic development remains less well defined. Here, we show that endothelial cells derived from the murine embryonic AGM region (AGM-EC) or fetal liver (FL-EC) provide an in vitro substrate for generation/maturation of HSC from HE/HSC-precursor populations derived from early stage murine embryos, which lack adult-engraftment capacity prior to co-culture. Notably, these EC substrates, endogenously expressing Notch ligands of the Jagged and Delta families, also support subsequent numerical expansion of AGM-derived HSC, expressing Notch1 and Notch2 receptors, as determined by limit dilution transplantation analysis. Consistent with a requirement for Notch activation in this process, phenotypic HSC expansion during EC co-culture is blocked by gamma-secretase mediated Notch pathway inhibition. Furthermore, we show that in vitro Notch activation by immobilized ligand Delta1, together with cytokines and inhibition of the TGF-beta pathway, is sufficient to increase the number of AGM-derived HSC in the absence of EC stroma. Expansion of phenotypic hematopoietic stem/progenitor cells generated by culture on Delta1 is inhibited by antibody-mediated blockade of the combination of Notch1 and Notch2, but not by either Notch1 or Notch2 alone. Consistent with this, Notch receptor-specific activation by either immobilized Notch1 or Notch2 antibody is sufficient to support AGM-derived HSC in vitro in preliminary experiments. Altogether, these studies suggest a role for Notch pathway activation by either Notch1 or Notch2 in supporting embryonic-stage HSC expansion subsequent to initial Notch1-mediated HSC specification.

Disclosures

Moon: Fate Therapeutics: co-founder Other. Rafii:Angiocrine Biosciences: Founder Other.
BCL-2 and c-MYC Rearrangements in Leukemic Phase of Diffuse Large B Cell Lymphoma Predicts Central Nervous System Involvement
Authors: Shuhua Yi , Zhong Shizheng , Zou Dehui , Li Chengwen , Li Zengjun , Liu Wei , Lv Rui , Zhang Peihong , Chen Huishu , Wang Huijun , Qiu Lugui
Published: October 2019

Introduction

Diffuse large B-cell lymphomas (DLBCL) are the most common lymphoid neoplasms but rarely with leukemic phase. Few studies had described the clinical characteristics of DLBCL with leukemic phase and found that these patients had high incidence of centre nervous system (CNS) involvement but with no knowledge of the reason.

Methods

We reviewed the clinical characteristics of 40 DLBCL patients in leukemic phase during 2001 and 2012. The DNA probes were LSI BCL2 and MYC Dual Color, Break Apart Rearrangement Probe were usedto detect the abnormalities of 18q21 and 8q24 by FISH.We also used LSI IGH/BCL2 Dual Color, Dual Fusion Translocation Probe and LSI IGH/MYC/CEP 8 Tri-Color Dual Fusion Probesto detect the partner fusion genes of BCL2 or MYC.

Results

The median age of these 40 patients was 46 years(range, 15-75) with 19 men patients. The median WBC was 12.32×109/l(range, 1.2-66.57)with a median 35.74% (range, 1–90) of circulating lymphomatous cells identified by flow cytometry.14 of these 40 patients (35%)had CNS involvement.There were gender differences between CNS involvement or not. Ten of the fourteen patients (71.4%) with CNS involvement were male, while in non CNS involvement group only 9 of 26 (34.6%) cases were male (p=0.046).57.1% patients with CNS involvement had IPI scores 4-5, which was higher than those without CNS involvement (11.5%, p=0.001).All of these 14 patients had elevated LDH was and was significantly higher than those without (61.5%, p=0.007).

There were 14 patients (33.5%) had the BCL2 or MYC aberrationrespectively and nine of them simultaneously had both of c-MYC and BCL2 rearrangement, which we defined as double-hit DLBCL patients here. No IGH/BCL2 fusion gene was detected and only one patient with IGH/MYC fusion with c-MYC rearrangement. Eight of the nine double-hit patients had CNS involvement, while only six of the other 31 patients with either BCL2 or MYC or none rearrangements had CNS involvement (88.9% vs. 19.4%; p＜0.001). By logistic regression analysis, we found that double-hit with BCL2 and c-MYC rearrangement was the only one independent factor correlated with CNS involvement with relative risk 15.3 (95% confidence interval, 1.4-171.1).

Conclusion

DLBCL patients in leukemic phase had higher incidence of CNS involvement and double-hit with BCL2 and c-MYC gene rearrangements might contribute to CNS involvement in this population.

Disclosures

No relevant conflicts of interest to declare.
B-Cell Receptors of Primary Central Nervous System Lymphoma Recognize Antigens in the Brain
Authors: Trepel Martin , Müller Fabian , Illerhaus Gerald , Glatzel Markus , Binder Mascha , Spies Elmar
Published: October 2019

INTRODUCTION: The pathobiology mediating the development of primary central nervous system lymphoma (PCNSL) and its exclusive manifestation in the brain and spinal cord is still poorly understood. The monoclonal B-cell receptor expressed in PCNSL cells have a germinal center typology, suggesting previous antigen contact and antigen-based selection during B-cell development. Furthermore, sequence analysis of B-cell receptors in various patients revealed a bias in the immunoglobulin repertoire in PCNSL, potentially indicating that distinct epitopes might elicit such an immune response. Nevertheless, the nature of potential antigens containing such epitopes is unknown. Here, we provide the first evidence that such antigens may be located within the central nervous system itself, which might contribute to the specific organ tropism of PCNSL.

METHODS: Cryopreserved tissue of five randomly chosen PCNSL cases and three control cases with mantle cell lymphoma were used for this study. The variable heavy and light chains of the B-cell receptors were amplified from extracted RNA and cloned in IgG1 expression vectors. Recombinant B-cell receptors were expressed and purified as soluble human immunoglobulins. To explore the potential recognition of central nervous system antigens, murine cryo- and paraffin-embedded brain tissue sections were immunostained using these PCNSL or mantle cell lymphoma-derived immunoglobulins as primary antibodies.

RESULTS: Four out of five recombinant B-cell receptors from PCNSL but none of the MCL-derived control immunoglobulins recognized distinct anatomical structures in the brain which resulted in unambiguous immunostaining especially in regions with high neuronal density. The staining of brain tissue was associated with neuronal cells in all four positive cases. Staining was stronger in the cerebellum and the hippocampus compared to other areas of the brain and staining intensity varied broadly among the four positive cases. In the cerebellum, all four positive B-cell receptors recognized the cytoplasm of Purkinje cells, the largest neurons in the brain (therefore, staining could be readily identified). Two of the B-cell receptors also recognized the Purkinje cell’s dendrites. Three of the PCNSL-derived immunoglobulins also stained the granule cells, the most frequent neuronal cell type in the brain. Two of those stained in the cytoplasm and one showed a patchy but strong staining pattern in the nucleus of these cells. All of these stainings including negative controls were reproduced multiple times in independent experiments.

CONCLUSION: B-cell receptors of primary central nervous system lymphomas recognize neuronal structures in the brain in an individually case-dependent pattern. This might point towards an important role of antigen recognition in the pathobiology and specific organ tropism of this lymphoma.

Disclosures

No relevant conflicts of interest to declare.
Rcor1 Deficiency Disrupts Myeloerythroid Lineage Differentiation and Causes Severe Myelodysplasia
Authors: Goldman Devorah C , Yao Huilan , Fan Guang , Mandel Gail , Fleming William H.
Published: October 2019

Previously, we showed that the co-repressor CoREST (Rcor1) is essential for the maturation of definitive erythroid cells in the mouse fetal liver. To elucidate Rcor1’s function in multilineage hematopoiesis in the adult, we conditionally deleted Rcor1 using Mx1-Cre. The loss of Rcor1 expression in hematopoietic cells led to the rapid onset of a severe anemia due to a complete block of erythropoiesis downstream of committed erythroid progenitors. By contrast, the production of megakaryocyte progenitors, megakaryocyte maturation and platelets were maintained. In the myelomonocytic lineages, although neutrophil differentiation was completely abrogated, the number of monocytic cells was significantly increased, resulting in a peripheral blood leukocytosis and monocytic infiltrations in the liver. Rcor1-deficient monocytes showed a 66% reduction in apoptosis and possessed ~100-fold more CFU-M activity than control cells. The CFU-M derived from Rcor1 deficient bone marrow could be serially replated up to 5 times; however, replating activity was entirely cytokine dependent. Defective myelomonocytic differentiation persisted following transplantation into wild type hosts for up to 12 months but did not progress to leukemia. To begin to understand at the molecular level how Rcor1 regulates monocyte expansion, we evaluated the expression levels of genes whose ectopic expression is associated with myeloid neoplasia. In Rcor1-deficient monocytes, Gata2, Meis1 and Hoxa9 were overexpressed by 8- to 200-fold. Taken together, these data demonstrate that Rcor1 is essential for both erythroid and myelomonocytic differentiation and that its loss of function causes significant myelodysplasia.

Disclosures

No relevant conflicts of interest to declare.
IL-17A-Mediated Notch Signaling in Multiple Myeloma
Authors: Prabhala Rao H. , Hideshima Teru , Fulciniti Mariateresa , Adamia Sophia , Bandi Rajya Lakshmi , Nanjappa Puru , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

Multiple myeloma (MM) is a plasma cell malignancy, however, significant abnormalities in T cell function are considered to provide help in uncontrolled growth and survival of MM cells. We have previously reported that IL-17A-producing Th17 cells are elevated in MM, that MM cells express IL-17 receptor, and IL-17A promotes MM cell growth and survival. We have reported that MM cells themselves produce IL-17A as confirmed by RT-PCR, Western blotting and immunostaining providing a possibility of both autocrine and paracrine role for IL-17A in MM. As Notch activation has been implicated in Th17 cell differentiation and IL17A production, we have here investigated the role of Notch pathway activation in IL-17A-mediated MM cell growth within the BM microenvironment. Notch consists of 4 proteins (1-4) and has 5 ligands (DLL-1,3,4 and jagged-1, 2). We analyzed RNA-Seq data from 117 newly-diagnosed MM patients and 18 normal plasma cells and observed high expression of Notch 1, and 2 and Notch target genes Hes-1 and Hey-1 but not Notch 3 and 4 in MM. For Notch 2, isoform 2 was highly predominant. Notch expression on MM cells was further confirmed by flow cytometric analysis (Notch1-84%, Notch2-86% and Notch3-3%). Evaluating functional role of Notch in MM, when MM cells were co-cultured with Notch ligand jagged 2-expressing 3T3 cells, IL-17A was able to further induce Notch target gene Hes-1 by 45%. Interestingly, increase in the expression of Notch 2 was also observed during this interaction (increased full-length protein by 65% and active intra-cellular protein by 145%). We next evaluated effect of both anti-IL-17 antibody and Notch inhibitors on MM cells. Anti-IL-17A monoclonal antibody inhibited full-length Notch2 protein expression by 54% and active intra-cellular protein by 85%, as determined by western blot analysis. The antibody inhibitory activity was confirmed with quantitative PCR. Importantly, IL-17A mAb inhibited Hes-1 protein expression by 83%. With the observed impact of Notch signaling in MM, we next evaluated notch inhibitors MRK003, and compound E, a γ-secretase inhibitors, to determine their impact on MM cell growth and survival. We observe that Notch inhibitors affect MM cell growth (inhibition by 43%%, N=5) and IL-6 production (inhibition by 60%, N=3) in co-culture with bone marrow stromal cells. These preclinical data establish the role of IL-17 as well as Notch signaling in myeloma and provides the rationale to evaluate anti-MM activity of anti-IL-17A monoclonal antibody and Notch inhibitors in MM.

Disclosures

No relevant conflicts of interest to declare.
Plasma Protease-Mediated Metabolism of Carfilzomib and Its Implications on Clinical Effects and Side Effects
Authors: Tristan Sissung M , Peer Cody J , Korde Neha , Nehal Lakhani , Sham Malinkody , Landgren Ola , Figg William D
Published: October 2019

Introduction: Similar to most other anti-cancer drugs, carfilzomib is administered at a defined dose per m2. Inspired by preclinical data showing that extrahepatic peptidases play an important role in the metabolism of carfilzomib, using samples from a prospective clinical trial, we conducted an investigation focusing on concentrations of albumin in peripheral blood and its effects on plasma protease-mediated metabolism of carfilzomib.

Methods: Baseline laboratory values, pharmacokinetics data, and response data were collected from the clinical trial. Minimal residual disease was determined by multi-parametric flow cytometry utilizing, 8-color flow panel and analyzes ≥ 3 x 106 events (sensitivity 1 x 10-5). To study the rate of metabolism in plasma, apheresis samples were obtained from the NIH blood bank, lyophilized albumin was added to plasma with and without a non-specific protease inhibitor cocktail (1X). Carfilzomib concentrations were obtained at several time points over the course of 24 hours by mass spectrometry via previously published methods.

Results: Patients with baseline serum albumin >4.0 g/dL had a near 4-fold greater clearance (1576 L/h vs 401.1 L/h; n=9 vs n=34) that was further increased in patients with normal white blood cell count. Consistent with the increase in clearance, 80% of patients (29/33) with albumin <4.0 g/dL became minimum residual disease (MRD) negative whereas only 44.4% of patients (4/9) with albumin >4.0 g/dL had a similar outcome. Therefore, individuals with baseline albumin >4.0 g/dL are at significantly greater risk for not responding to the combination of carfilzomib, lenalidomide, and dexamethasone [OR (95%CI) = 7.3 (1.4-36.7); P=0.0049]. Median progression-free survival was also longer in patients with albumin <4.0 g/dL vs. albumin >4.0 g/dL (unreached vs. 18.8 months; P=0.0002). A trend between albumin and lymphopenia was also detected (P=0.17). The addition of 1.0 g/dL albumin increased the ex vivo proteolytic rate of carfilzomib in apheresis plasma from healthy individuals (74% vs 57.2% unreacted carfilzomib after 24 hours at 37°C), and the addition of a non-specific protease inhibitor reduced carfilzomib metabolism in plasma

Conclusions: Although they have lower grade myeloma, patients with albumin >4.0 g/dL, surprisingly, are at greater risk at having worse clinical outcomes to carfilzomib therapy; this finding appears to depend upon the ability of albumin to directly potentiate plasma protease-mediated metabolism of carfilzomib. Therefore, dose adjustments or concomitant therapy with protease inhibitors may be warranted in such patients. Efforts are underway to identify specific proteases that metabolize carfilzomib and determine potential inhibitors that may be clinically useful.

Disclosures

No relevant conflicts of interest to declare.
Two-Track Epigenetic Remodeling and Backtracking to Embryonic Stem Cell Bivalency in B-Cell Acute Lymphoblastic Leukemias
Authors: Lee Seung-Tae , Muench Marcus O. , Fomin Marina , Xiao Jianqiao , Zhou Mi , de Smith Adam J. , Martín-Subero José , Heath Simon , Houseman E. Andres , Roy Ritu , Wrensch Margaret R. , Wiencke John K. , Metayer Catherine , Wiemels Joseph L.
Published: October 2019

Purpose: We sought to define the drivers and functional consequences of DNA methylation changes in childhood pre-B cell leukemia. A comprehensive analysis of such DNA methylation changes will reveal aspects of the etiology and pathogenesis of leukemias, and suggest potential therapeutic modalities.

Background: The epigenome is extensively altered in carcinogenesis, but the proximate causes, functional consequences, and overall patterning of DNA methylation changes in the pediatric leukemias is not well defined.

Methods: We bisulfite sequenced at single base pair resolution two common pre-B cell leukemia case DNAs and a pre-B cell control (CD19+/CD34+ pre-B cells from normal marrow), and analyzed DNA methylation by high definition microarray in another 227 subjects. RNA expression was available for 82 samples, including the sequenced pre-B cells and leukemias.

Results: Epigenetic alteration of B-ALLs occurred in two tracks: de novo methylation of small functional compartments and demethylation of large inter-compartmental backbones. The de novo methylation occurred preferentially at polycomb targets and binding sites for the transcriptional co-repressor CtBP2. DNA methylation of ETV6-AML1 leukemia was overall equivalent to the control, with high hyperdiploid methylation levels being 4.4% lower. Hierarchical clustering revealed four groups, with ETV6-AML1 and hyperdiploid leukemias enriched within two separate groups. DNA methylation deviations were exaggerated in lamina-associated domains, with differences corresponding to these methylation clusters and/or cytogenetic groups. DNA methylation changes were correlated with gene expression changes, and a key polycomb expression signature was reversed when DNA methylation marks were pharmacologically removed in leukemia cell lines.

Discussion: While leukemia cells retain the majority of their developmentally determined DNA methylation patterns, key modifications with leukemogenesis revealed both broad patterns and functional targets with some dependence on known characteristics of pre-B cell leukemias. Our data suggested a pivotal role of polycomb and CTBP2 in de novo methylation, which may be traced back to bivalency status of embryonic stem cells. Driven by these potent epigenetic modulations, suppression of polycomb target genes was observed along with disruption of developmental fate and cell cycle and mismatch repair pathways and altered activities of key upstream regulators. An appreciation and validation of the observed changes will lead to new insights into etiology, pathogenesis, and treatments of childhood ALL.

Disclosures

No relevant conflicts of interest to declare.
Rituximab May be Not Suitable for Epstein Barr Virus-Induced Hemophagocytic Lymphohistiocytosis in Chinese
Authors: Wang Zhao , Wang Yini , Wei Na , Zeng Xiangzong , Huang Wenqiu , Wang Jingshi
Published: October 2019

Purpose: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome. Epstein-Barr virus (EBV) is the most frequent infection associated with HLH. Some studies revealed that rituximab containing regimens is an effective treatment for EBV- HLH. But we didn’t make the similar observation in Chinese adult patients. This study was aim to describe the outcomes and tried to explain the possible causes.

Methods: We performed an investigation involving 46 EBV-HLH patients. Eleven of them received treatment with rituximab plus HLH-94 protocol. The other 35 patients were only treated with HLH-94 protocol. The EBV-DNA load and survive rate of patients were used to evaluated curative effective. We identified the lineage of EBV-infected cell by immunohistochemistry (ICH) in combination with in site hybridization (ISH) in Epstein-barr Virus Encoded Rnas (EBER) for cell type-specific markers.

Result: The eleven patients received 3 rituximab infusions on average (range 2-4) at a dosage of 375mg/m2 every week. We found no significant difference in EBV-DNA load between the rituximab containing regimens group and conventional HLH-94 group at diagnosis (median load was 3.2×105copies/ml vs 2.8×105copies/ml) and 4 weeks after therapy (median load 6.4×103copies/ml vs 5.5×103copies/ml). The survive rates of the two groups on 2 months, 6 months and 12 months respectively were 45.5% vs 42.9%, 27.3% vs 28.6%, 18.2% vs 20.0%. The pathological examination of tissue biopsy in the patients from rituximab containing regimens group revealed that EBER was positive in all patients. We identified the lineage of EBV-infected cell by double staining——ICH in combination with ISH. Nine of them were CD3+/EBER+, and only two of them were CD20+/EBER+.

Conclusion: It suggested that rituximab may be not suitable for EBV-HLH patients in China. Most probably, EBV predominantly infects T cells in Chinese, which could explain the nonsuperiority of rituximab in Chinese adult EBV-HLH. It is necessary to make further research to demonstrate it.

Disclosures

No relevant conflicts of interest to declare.
The Tetraspanin CD81 Facilitates Tumor Metastasis By Modulating Immune Suppression
Authors: Vences Catalan Felipe , Rajapaksa Ranjani , Srivastava Minu K. , Marabelle Aurelien , Levy Shoshana
Published: October 2019

Tumor metastasis is the major cause of cancer mortality. While immune cells are aimed at halting metastases, the proliferating cancer cells at the primary tumor site recruit cells that counteract the adaptive anti-tumor immune response. Indeed, it is well established that tumors induce the accumulation and proliferation of immune suppressive cells, such as regulatory T cells (Tregs) and Myeloid Derived Suppressor cells (MDSC).

The tetraspanin CD81 belongs to a family of proteins that play an important role in the immune system as evident by impaired immune functions of DCs, B and T cells in several tetraspanin-deficient mice (CD37, CD81, CD151, and CD82).

While many studies have addressed the function of CD81 in infection and in the immune system, few have focused on its role in tumorigenesis and metastasis. Here we report that the growth of subcutaneously implanted breast cancer tumors (4T1) is slightly impaired in CD81 knockout (CD81KO) compared to wild type mice. Moreover, CD81KO mice develop very few lung metastases compared to their wild type and heterozygous counterparts.

Both wild type and CD81KO tumor-bearing mice show substantial increases in Tregs, and MDSCs. However, these Tregs and MDSCs differ functionally - those derived from wild type mice are effective suppressors of T cell proliferation. By contrast, Tregs and MDSCs derived from tumor-bearing CD81KO mice do not suppress the proliferation of Teff cells. Thus, it is highly likely that while Teff cells are greatly restrained by Tregs and MDSCs in wild type tumor-bearing mice, the impairment of both suppressive cell populations in CD81KO mice enables the anti-tumor function thereby opposing metastases.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.